JP2004506878A - HCV antigen / antibody combination assay - Google Patents

Abstract

The present invention provides a combination assay for HCV core antigen and NS3 / 4a antibody. This HCV core antigen and NS3 / 4a antibody combination assay can detect both HCV antigen and HCV antibodies present in a sample using a single solid matrix. In addition, the present invention also provides an immunoassay solid support for use in this HCV core antigen and NS3 / 4a antibody combination assay.

Description

^＊ＨＣＶ−１に対して番号付けられている。Ｃｈｏｏら（１９９１）Ｐｒｏｃ．Ｎａｔｌ．Ａｃａｄ．Ｓｃｉ．ＵＳＡ８８：２４５１−２４５５を参照のこと。
【００７６】
複数のＨＣＶ抗原は、鎖が天然には存在しないアミノ酸の単一の連続した鎖の一部である。従って、エピトープの線形順序は、エピトープが生じるゲノムにおけるそれらの線形順序とは異なる。本明細書中における使用のためのＭＥＦＡの配列の線形順序は、好ましくは、最適な抗原性のために配置される。好ましくは、これらのエピトープは、１つより多いＨＣＶ株由来であり、従って、１つのアッセイで複数のＨＣＶ株を検出する付加能力を提供する。従って、本明細書中における使用のためのＭＥＦＡは、上記のポリタンパク質由来の様々な免疫原性領域を含み得る。さらに、このポリタンパク質のコア領域におけるフレームシフトから生じるタンパク質（例えば、国際公開番号ＷＯ９９／６３９４１に記載されるようなタンパク質）は、ＭＥＦＡにおいて使用され得る。所望ならば、ＨＣＶポリタンパク質由来の１つ以上のエピトープの少なくとも２、３、４、５、６、７、８、９もしくは１０、またはそれ以上は、融合タンパク質中で生じ得る。
【００７７】
例えば、Ｅ２の超可変領域（例えば、アミノ酸３８４〜４１０または３９０〜４１０にわたる領域）由来のエピトープは、ＭＥＦＡ抗原に含まれ得る。特に有効なＥ２エピトープは、この領域由来のコンセンサス配列（例えば、コンセンサス配列Ｇｌｙ−Ｓｅｒ−Ａｌａ−Ａｌａ−Ａｒｇ−Ｔｈｒ−Ｔｈｒ−Ｓｅｒ−Ｇｌｙ−Ｐｈｅ−Ｖａｌ−Ｓｅｒ−Ｌｅｕ−Ｐｈｅ−Ａｌａ−Ｐｒｏ−Ｇｌｙ−Ａｌａ−Ｌｙｓ−Ｇｌｎ−Ａｓｎ（これは１型ＨＣＶゲノムのアミノ酸３９０〜４１０のコンセンサス配列を表す））を含むエピトープである。本発明のＭＥＦＡに存在する代表的なＥ２エピトープは、アミノ酸３９０〜４４４にわたるハイブリッドエピトープを含み得る。このようなハイブリッドＥ２エピトープは、ＨＣＶ　Ｅ２のアミノ酸４１１〜４４４のネイティブのアミノ酸配列に融合したアミノ酸３９０〜４１０を表すコンセンサス配列を含み得る。
【００７８】
さらに、抗原は、様々なＨＣＶ株由来であり得る。ＨＣＶの複数のウイルス株が公知であり、そしてこれらの株のいずれか由来のエピトープは、融合タンパク質において使用され得る。生物体の任意の所定の種は、個々の生物体ごとに異なり、さらにウイルスのような所定の生物体は多数の異なる株を有することが周知である。例えば、上で説明されたように、ＨＣＶは、少なくとも６個の遺伝型を含む。これらの遺伝型の各々は、等価な抗原決定基を含む。より詳細には、各株は、ウイルスの全ての株に存在するが、ウイルス株ごとにわずかに異なる多数の抗原決定基を含む。例えば、ＨＣＶは、５−１−１として公知の抗原決定基を含む（図１を参照のこと）。この特定の抗原決定基は、ＨＣＶの３つの異なるウイルス株上で３つの異なる形態で現れる。従って、本発明の好ましい実施形態において、５−１−１の３つ全ての形態は、目的のイムノアッセイにおいて使用される多エピトープ融合抗原上に現れる。同様に、異なるＨＣＶ株のコア領域由来の等価な抗原決定基がまた存在し得る。一般的に、等価な抗原決定基は、アミノ酸配列に関して高度な相同性を有し、この相同性の程度は、整列された場合、一般に３０％以上、好ましくは４０％以上である。本発明の多コピーエピトープはまた、同じエピトープの正確なコピーである複数のコピーを含み得る。
【００７９】
本発明のアッセイと共に使用するための代表的なＭＥＦＡは、国際公開番号ＷＯ９７／４４４６９に記載される。本明細書中で使用するためのさらなる代表的なＭＥＦＡとしては、ＭＥＦＡ１２、ＭＥＦＡ１３、およびＭＥＦＡ１３．１と呼ばれるＭＥＦＡが挙げられる。これらのＭＥＦＡは、単に例示であり、そしてＨＣＶゲノム由来の他のエピトープもまた本発明のアッセイを用いる用途を見出し、そしてこれらまたは他のＭＥＦＡに組み込まれえることが理解されるべきである。
【００８０】
ＭＥＦＡ１２のＤＮＡ配列および対応するアミノ酸配列を、図７Ａ〜７Ｆに示す。ＭＥＦＡ１２の一般構造式は、図６に示され、そして以下のとおりである：ｈＳＯＤ−Ｅ１（タイプ１）−Ｅ２　ＨＶＲコンセンサス（タイプ１ａ）−Ｅ２　ＨＶＲコンセンサス（タイプ１および２）−ｃ３３ｃショート（タイプ１）−５−１−１（タイプ１）５−１−１（タイプ３）−５−１−１（タイプ２）−ｃ１００（タイプ１）−ＮＳ５（タイプ１）−ＮＳ５（タイプ１）−コア（タイプ１＋２）−コア（タイプ１＋２）。この多コピーエピトープは、ＨＣＶ−１に関して番号付けされた以下のアミノ酸配列を含む（以下に記載されるアミノ酸配列の番号付けは、Ｃｈｏｏら（１９９１）Ｐｒｏｃ．Ｎａｔｌ．Ａｃａｄ．Ｓｃｉ．ＵＳＡ　８８：２４５１−２４５５で提供される番号付けの指示に従い、ここでアミノ酸＃１は、コア領域のコード配列によりコードされる１番目のメチオニンである）：スーパーオキシドジスムターゼ（ＳＯＤ、タンパク質の組換え発現を促進するために使用される）のアミノ酸１〜６９；Ｅ１領域由来のポリタンパク質のアミノ酸３０３〜３２０；ＨＣＶ−１ａ　Ｅ２の超可変領域のコンセンサス配列を表すポリタンパク質のアミノ酸３９０〜４１０；ＨＣＶ−１およびＨＣＶ−２のＥ２超可変領域のコンセンサス配列を表す、領域Ｅ２由来のポリタンパク質のアミノ酸３８４〜４１４；ヘリカーゼを規定するＨＣＶ−１ポリタンパク質のアミノ酸１２１１〜１４５７；５−１−１のアミノ酸１６８９〜１７３５由来のエピトープの３つのコピー（２つはＨＣＶ−１由来であり、１つはＨＣＶ−３由来であり、そして１つはＨＣＶ−２由来であり、これらのコピーは、ＨＣＶの３つの異なるウイルス株由来の等価な抗原決定基である）；ＨＣＶ−１のＨＣＶポリペプチドＣ１００である、ポリタンパク質のアミノ酸１９０１〜１９３６；ＨＣＶ−１のＮＳ５領域由来のエピトープの２つの正確なコピー（この各々はＨＣＶポリタンパク質のアミノ酸２２７８〜２３１３を有する）；およびコア領域由来の３つのエピトープの２つのコピー（１つはＨＣＶ−１由来であり、１つはＨＣＶ−２由来であり、これらのコピーは、ＨＣＶ−１のアミノ酸９〜５３および６４〜８８、ならびにＨＣＶ−２のアミノ酸６７〜８４により表される等価な抗原決定基である）。
【００８１】
表２は、本明細書中の図７Ａ〜７Ｆを参照して、ＭＥＦＡ１２中の様々なエピトープのアミノ酸位置を示す。この表における番号付けは、ＨＣＶ−１に関する。Ｃｈｏｏら（１９９１）Ｐｒｏｃ．Ｎａｔｌ．Ｓｃｉ．ＵＳＡ　８８：２４５１−２４５５を参照のこと。ＭＥＦＡ１３および１３．１はまた、それぞれ、表３および４に示されるような改変を有する、ＭＥＦＡ１２について上で特定された一般式を共有する。
【００８２】
【表２】

＊ＳＯＤタンパク質は、検出結合体であるＨＲＰ標識抗ＳＯＤ抗体がＭＥＦＡに結合しないように切断される。コアエピトープは、検出に使用されるＨＣＶコアに対する抗体がＭＥＦＡに結合しないように変異される。
【００８３】
【表３】

＊５−１−１エピトープは、ＮＳ３／４ａ組換えタンパク質によって標的化される可能な切断部位（ＣＳまたはＣＡ）を除去することによって改変される。ＣＳまたはＣＡの代わりに、この配列はＰＩに変えられる。さらに、ＳＯＤタンパク質は、検出結合体であるＨＲＰ標識抗ＳＯＤ抗体がＭＥＦＡに結合しないように変異される。コアエピトープは、検出に使用されるＨＣＶに対する抗体がＭＥＦＡに結合しないように変異される。
【００８４】
【表４】

＊５−１−１エピトープは、ＮＳ３／４ａ組換えタンパク質によって標的化される可能な切断部位（ＣＳまたはＣＡ）を除去することによって改変される。ＣＳまたはＣＡの代わりに、この配列はＰＩに変えられる。さらに、ＳＯＤタンパク質は、検出結合体であるＨＲＰ標識抗ＳＯＤ抗体がＭＥＦＡに結合しないように変異される。コアエピトープは、検出に使用されるＨＣＶコアに対する抗体がＭＥＦＡに結合しないように変異される。
【００８５】
１つのアッセイ様式において、サンプルは、以下にさらに記載されるように、固体支持体と合わされる。サンプルがＨＣＶに感染している場合、コア抗原およびこの固体支持体上に存在するこれらのエピトープに対するＨＣＶ抗体は、固体支持体成分に結合する。次いで、検出可能に標識された抗コア抗体が、加えられる。この標識された抗コア抗体は、固体支持体に結合する抗コア抗体とは異なるエピトープに対して指向される。この抗コア抗体は、固体支持体上の抗コア抗体により捕捉されるコア抗原に結合する。
【００８６】
生物学的サンプル由来の捕捉されたＨＣＶ抗体（この捕捉されたサンプルのＨＣＶ抗体はＮＳ３／４ａエピトープと反応性である）と反応する抗原もまた加えられる。この抗原は、好ましくは、ＨＣＶポリタンパク質のＮＳ３領域由来のエピトープである。この抗原は、サンプル由来の捕捉されたＨＣＶ抗体に結合する。このようなエピトープを含む多数の抗原が公知であり、これには、ｃ３３ｃおよびｃ１００領域由来の抗原、およびＮＳ３エピトープ（例えば、ｃ２５）を含む融合タンパク質が挙げられるが、これらに限定されない。これらおよび他のＮＳ３エピトープは、本発明のアッセイにおいて有用であり、そして当該分野で公知であり、そして例えば、以下に記載されている：Ｈｏｕｇｈｔｏｎら、米国特許第５，３５０，６７１号；Ｃｈｉｅｎら、Ｐｒｏｃ．Ｎａｔｌ．Ａｃａｄ．Ｓｃｉ．ＵＳＡ（１９９２）８９：１００１１−１００１５；Ｃｈｉｅｎら、Ｊ．Ｇａｓｔｒｏｅｎｔ．Ｈｅｐａｔｏｌ．（１９９３）８：Ｓ３３−３９；Ｃｈｉｅｎら、国際公開番号ＷＯ９３／００３６５；Ｃｈｉｅｎ，Ｄ．Ｙ．，国際公開番号ＷＯ９４／０１７７８；ならびに共有に係る特許になった米国特許出願番号０８／４０３，５９０および同０８／４４４，８１８。
【００８７】
上記の抗原に対する第２の標識された抗体が加えられる。この抗体は、抗原に含まれる任意のエピトープに対して指向され得る。例えば、この抗体は、抗原中に存在するＮＳ３領域に対して指向され得る。あるいは、上記の抗原が融合タンパク質として発現される場合、第２の標識された抗原は、融合パートナーに対して指向され得る。特に、固体支持体がＭＥＦＡを含む場合、さらなる抗原および抗体がアッセイに加えられ得る。これらのアッセイ様式は、以下にさらに説明される。
【００８８】
本発明の代表的なアッセイを図２に示す。この図に示されるように、固体支持体は、２つの抗コアモノクローナル抗体（ｃ１１−３およびＣ１１−７と呼ばれる）を含む。これらの抗体は、アミノ酸１０〜５３（ＨＣＶ１ポリタンパク質配列に関して番号付けした）のコアタンパク質のＮ末端領域に見出されるエピトープに対して指向される。この固体支持体はまた、ＮＳ３／４ａに対するエピトープを含む。生物学的サンプルが、この固体支持体に加えられる。ＨＣＶコア抗原、およびＮＳ３／４ａエピトープに対する抗体（この両方はサンプル中に存在する）は、固体支持体上の捕捉試薬に結合する。
【００８９】
西洋ワサビペルオキシダーゼ（ＨＲＰ）−標識抗コアモノクローナル抗体ｃ１１−１４（アミノ酸の１２０〜１３０位に見出されるコアのＣ末端領域に対する抗体、ＨＣＶ１ポリタンパク質配列に関して番号付けされている）が、加えられる。ヒトＳＯＤ（ｈＳＯＤ）由来の配列およびｃ３３ｃ領域由来のエピトープを含む融合タンパク質が、この融合タンパク質のＳＯＤ部分に対する第２のＨＲＰ標識抗体と同様に、加えられる。ＳＯＤ−ｃ３３ｃ融合タンパク質は、抗ＮＳ３抗体に結合し、そして抗ＳＯＤ抗体は、次いで、ＳＯＤ−ｃ３３ｃ融合タンパク質に結合する。標識の検出は、ＨＣＶ感染の存在を表す。
【００９０】
本発明の別の代表的なアッセイを図８に示す。抗体アッセイの構成は、ＮＳ３／４ａおよびＭＥＦＡ１２の両方を使用する抗原−抗体−抗原サンドイッチ捕捉アッセイである。固体支持体は、上記の２つの抗コアモノクローナル抗体、ＮＳ３／４ａに対するエピトープ、および代表的なＭＥＦＡであるＭＥＦＡ１２（これは、ヒトＳＯＤの短縮型を含む）を含む。上記のアッセイのように、生物学的サンプルがこの固体支持体に加えられる。ＨＣＶコア抗原、ならびにＮＳ３／４ａエピトープおよびＭＥＦＡのエピトープに対する抗体は、サンプル中に存在し、これは固体支持体上の捕捉試薬に結合する。２つの抗原（１つは、ＮＳ３／４ａに結合するサンプル抗体（上記のような）と反応性であり、そして１つは、ＭＥＦＡ１２に結合するサンプル抗体と反応性である）が添加される。図８において、ＭＥＦＡ１２／サンプル抗体複合体と反応性の抗原は、ＳＯＤ分子とｃ２２ｋｓΔ４７−Ｌ４４Ｗとの間の融合物である。このｃ２２ｋｓ抗原はコア領域由来であり、そしてポリタンパク質のアミノ酸Ｌｙｓ_１０〜Ｓｅｒ_９９、ならびに通常存在するＡｒｇ_４７の欠失、および４４位におけるＬｅｕのＴｒｐでの置換を含む。抗原検出結合体は、上記の第２のＨＲＰ標識モノクローナル抗ＳＯＤ抗体である。
【００９１】
上記の抗原／抗体組み合わせアッセイは、ＨＣＶコア抗原ならびにＮＳ３／４ａおよび／またはコアに対する抗体の両方が、同じアッセイにおいて同じ支持体により検出され得る場合、特に有利である。さらに、上記のように、さらなるＨＣＶエピトープ（例えば、ｃ１００、５−１−１、ＮＳ５抗原に対するＳＯＤ融合タンパク質）、およびポリタンパク質のコア領域におけるフレームシフトから生じるタンパク質（例えば、国際公開番号ＷＯ９９／６３９４１に記載されるようなタンパク質）は、ＨＣＶの他の非構造的エピトープを網羅する組み合わせカクテルにおいて使用され得る。
【００９２】
本発明のさらなる理解のために、より詳細な議論は、本発明のイムノアッセイにおける使用のための抗体の生成、このイムノアッセイにおける使用のためのポリペプチドの生成、およびこのイムノアッセイを実施する方法に関して以下に記載される。
【００９３】
（ＨＣＶイムノアッセイにおける使用のための抗体の生成）
上で説明したように、アッセイは、固体支持体（例えば、１つ以上の抗コア抗体）に結合した種々の抗体、およびＨＣＶ感染がサンプル中に存在する場合に形成される抗原／抗体複合体を検出する種々の抗体を利用する。これらの抗体は、ポリクローナル抗体調製物またはモノクローナル抗体調製物、単一特異的抗血清、ヒト抗体であってもよいし、例えば、ヒト化抗体、変性抗体、Ｆ（ａｂ’）_２フラグメント、Ｆ（ａｂ）フラグメント、Ｆｖフラグメント、単一ドメイン抗体、二量体または三量体の抗体フラグメント構築物、ミニ抗体（ｍｉｎｉｂｏｄｉｅｓ）、または問題の抗原に結合するそれらの機能的フラグメントのような抗体ハイブリッド抗体またはキメラ抗体であってもよい。
【００９４】
抗体は、当業者に周知の技術、ならびに例えば、米国特許第４，０１１，３０８号；同第４，７２２，８９０号；同第４，０１６，０４３号；同第３，８７６，５０４号；同第３，７７０，３８０号；および同第４，３７２，７４５号に記載される技術を使用して生成される。例えば、ポリクローナル抗体は、適切な動物（例えば、マウス、ラット、ウサギ、ヒツジまたはヤギ）を、目的の抗原で免疫することによって生成される。免疫原性を増強するために、抗原は、免疫の前にキャリアに結合され得る。このようなキャリアは、当業者に周知である。免疫は、一般に、生理食塩水中、好ましくは、フロイントの完全アジュバントのようなアジュバント中の抗原を混合または乳化することによって、そして混合物またはエマルジョンを非経口的（一般に、皮下または筋肉内）に注射することによって行なわれる。動物は、一般に、好ましくは、フロイントの不完全アジュバントを使用して、生理食塩水中の抗原の１回以上の注射で、２〜６週後に追加免疫される。抗体はまた、当該分野で公知の方法を使用する、インビトロ免疫によって生成され得る。次いで、ポリクローナル抗血清は、免疫動物から得られる。例えば、抗ＨＣＶポリクローナル抗体の生成については、Ｈｏｕｇｈｔｏｎら、米国特許第５，３５０，６７１号を参照のこと。
【００９５】
モノクローナル抗体は、一般に、ＫｏｈｌｅｒおよびＭｉｌｓｔｅｉｎ（１９７５）Ｎａｔｕｒｅ　２５６：４９５−４９７の方法またはその改変物を使用して調製される。代表的には、マウスまたはラットが、上記のように免疫される。しかし、血清を抽出するために動物を出血させるのではなく、脾臓（および必要に応じていくつかの大きいリンパ節）が取り出され、そして単一の細胞に分離される。所望の場合、脾臓細胞は、抗原でコーティングしたプレートまたはウェルに細胞懸濁液を適用することによって、（非特異的に接着した細胞の除去後に）スクリーニングされ得る。Ｂ細胞（抗原に特異的な膜結合免疫グロブリンを発現する）はプレートに結合し、そして懸濁液の残りと共に洗い流されない。次いで得られたＢ細胞（または全ての分離した脾臓細胞）は、黒色腫細胞と融合するように誘導されてハイブリドーマを形成し、そして選択的な培地（例えば、ヒポキサンチン、アミノプテリン、チミジン培地、「ＨＡＴ」）で培養される。得られたハイブリドーマは、限界希釈によってプレートされ、そして、免疫抗原に特異的に結合する（そして関連していない抗原に結合しない）抗体の生成についてアッセイされる。次いで、選択されたモノクローナル抗体スクリーニングハイブリドーマは、インビトロ（例えば、組織培養瓶または中空線維反応器中）またはインビボ（例えば、マウスの腹水のような）のいずれかで培養される。
【００９６】
種々の抗ＨＣＶモノクローナル抗体の生成は、例えば、Ｈｏｕｇｈｔｏｎら、米国特許第５，３５０，６７１号；Ｃｈｉｅｎら、国際公開番号ＷＯ９３／００３６５；共有に係る、特許となった米国特許出願番号０８／４０３，５９０および同０８／４４４，８１８；およびＫａｓｈｉｗａｋｕｍａら、米国特許番号５，８７１，９０４号において記載されている。
【００９７】
上に説明されたように、目的の抗原を認識する能力を保持する抗体フラグメントはまた、目的のイムノアッセイにおいて用途を見出す。インタクトな抗体分子の免疫学的結合特性を示し得る抗原結合部位を含む多数の抗体フラグメントが公知である。例えば、機能的な抗体フラグメントは、例えば、ペプシンを使用して、抗体分子から抗原結合を担わない定常領域を切断することによって生成され、Ｆ（ａｂ’）_２フラグメントを生成する。これらのフラグメントは、２つの抗原結合部位を含むが、重鎖の各々から定常領域の一部分を欠く。同様に、所望な場合、Ｆａｂフラグメント（単一の抗原結合部位を含む）が、例えば、パパインでのポリクローナル抗体またはモノクローナル抗体の消化によって、生成され得る。機能的フラグメント（重鎖および軽鎖の可変領域のみを含む）は、免疫グロブリン分子の組換え生成または優先的タンパク質分解切断のような標準的な技術を使用して、生成され得る。これらのフラグメントは、Ｆｖとして公知である。例えば、Ｉｎｂａｒら（１９７２）Ｐｒｏｃ．Ｎａｔ．Ａｃａｄ．Ｓｃｉ．ＵＳＡ　６９：２６５９−２６６２；Ｈｏｃｈｍａｎら（１９７６）Ｂｉｏｃｈｅｍ　１５：２７０６−２７１０；およびＥｈｒｌｉｃｈら（１９８０）Ｂｉｏｃｈｅｍ　１９：４０９１−４０９６を参照のこと。
【００９８】
単鎖Ｆｖ（「ｓＦｖ」または「ｓｃＦｖ」）ポリペプチドは、ペプチドコードリンカーによって連結されるＶ_Ｈコード遺伝子およびＶ_Ｌコード遺伝子を含む遺伝子融合物から発現される共有結合したＶ_Ｈ−Ｖ_Ｌヘテロ二量体である。Ｈｕｓｔｏｎら（１９８８）Ｐｒｏｃ．Ｎａｔ．Ａｃａｄ．Ｓｃｉ．ＵＳＡ　８５：５８７９−５８８３。天然に凝集しているが化学的に分離した、抗体Ｖ領域由来の軽鎖および重鎖ポリペプチドを、抗原結合部位の構造に実質的に類似した三次元構造に折り畳まれるｓＦｖ分子に変換する化学的構造（リンカー）を区別および開発するための多数の方法が記載されている。例えば、米国特許第５，０９１，５１３号、同第５，１３２，４０５号、および同第４，９４６，７７８号を参照のこと。ｓＦｖ分子は、当該分野で記載される方法を使用して生成され得る。例えば、Ｈｕｓｔｏｎら（１９８８）Ｐｒｏｃ．Ｎａｔ．Ａｃａｄ．Ｓｃｉ．ＵＳＡ　８５：５８７９−５８８３；米国特許第５，０９１，５１３号、同第５，１３２，４０５号、および同第４，９４６，７７８号を参照のこと。設計基準は、一方の鎖のＣ末端と、他方の鎖のＮ末端との間の距離にわたる適切な長さを決定することを包含し、ここで、リンカーは、一般に、コイル化する傾向がないか、または二次構造を形成しない親水性低分子アミノ酸残基から形成される。このような方法は、当該分野で公知である。例えば、米国特許第５，０９１，５１３号、同第５，１３２，４０５号、および同第４，９４６，７７８号を参照のこと。適切なリンカーは、一般に、代替のセットのグリシンおよびセリン残基のポリペプチド鎖を含み、そして可溶性を増強するために、挿入されたグルタミン酸およびリジン残基を含み得る。
【００９９】
「ミニ抗体（ｍｉｎｉ−ａｎｔｉｂｏｄｉｅｓ）」または「ミニ抗体（ｍｉｎｉｂｏｄｉｅｓ）」もまた、本発明を用いる用途が見出される。ミニ抗体は、ヒンジ領域によってｓＦｖから分離された、そのＣ末端にオリゴマー形成ドメインを含むｓＦｖポリペプチド鎖である。Ｐａｃｋら（１９９２）Ｂｉｏｃｈｅｍ　３１：１５７９−１５８４。オリゴマー形成ドメインは、さらなるジスルフィド結合によってさらに安定化され得る自己会合αヘリックス（例えば、ロイシンジッパー）を含む。このオリゴマー形成ドメインは、膜を横切る特定の方向への折り畳みと適合性であるように設計され、この折り畳みは、ポリペプチドの機能的な結合タンパク質へのインビボの折り畳みを容易にすると考えられているプロセスである。一般に、ミニ抗体は、当該分野で周知の方法を使用して生成される。例えば、Ｐａｃｋら（１９９２）Ｂｉｏｃｈｅｍ　３１：１５７９−１５８４；Ｃｕｍｂｅｒら（１９９２）Ｊ　Ｉｍｍｕｎｏｌｏｇｙ　１４９Ｂ：１２０−１２６を参照のこと。
【０１００】
（ＨＣＶイムノアッセイにおける使用のための抗原の生成）
上で説明したように、本発明の分子は、一般に、組換え的に生成される。従って、本発明と共に使用するための、ＨＣＶ抗原をコードするポリヌクレオチドは、分子生物学の標準的な技術を使用して作製され得る。例えば、上記の分子をコードするポリヌクレオチド配列は、組換え方法を使用して（例えば、この遺伝子を発現する細胞由来のｃＤＮＡおよびゲノムライブラリをスクリーニングすることによって、またはこの遺伝子を含むことが知られているベクターから遺伝子を誘導することによって）得られ得る。さらに、所望の遺伝子は、当該分野（例えば、Ｈｏｕｇｈｔｏｎら、米国特許第５，３５０，６７１号）に記載される技術を使用して、ウイルス核酸分子から直接単離され得る。目的の遺伝子はまた、クローン化よりむしろ合成的に生成され得る。これらの分子は、特定の配列について適切なコドンを用いて設計され得る。次いで、完全配列は、標準的な方法により調製される重複オリゴヌクレオチドから構築され、そして完全コード配列に構築される。例えば、Ｅｄｇｅ（１９８１）Ｎａｔｕｒｅ　２９２：７５６；Ｎａｍｂａｉｒら（１９８４）Ｓｃｉｅｎｃｅ　２２３：１２９９；およびＪａｙら（１９８４）Ｊ．Ｂｉｏｌ．Ｃｈｅｍ．２５９：６３１１を参照のこと。
【０１０１】
従って、特定のヌクレオチド配列は、所望の配列を保有するベクターから得られ得るか、または当該分野で公知の様々なオリゴヌクレオチド合成技術（例えば、適切な場合、部位特異的変異誘発およびポリメラーゼ連鎖反応（ＰＣＲ））を使用して、完全にもしくは部分的に合成され得る。例えば、Ｓａｍｂｒｏｏｋ、（前出）を参照のこと。特に、所望の配列をコードするヌクレオチド配列を得る１つの方法は、従来の自動化ポリヌクレオチド合成機で生成される重複合成オリゴヌクレオチドの相補的なセットをアニーリングし、続いて適切なＤＮＡリガーゼで連結し、そしてＰＶＲによりこの連結したヌクレオチド配列を増幅することによるものである。例えば、Ｊａｙａｒａｍａｎら、（１９９１）Ｐｒｏｃ．Ｎａｔｌ．Ａｃａｄ．Ｓｃｉ．ＵＳＡ　８８：４０８１〜４０８８を参照のこと。さらに、オリゴヌクレオチド指向型合成（Ｊｏｎｅｓら（１９８６）Ｎａｔｕｒｅ　５４：７５〜８２）、既存のヌクレオチド領域のオリゴヌクレオチド指向型変異誘発（Ｒｅｉｃｈｍａｎｎら（１９８８）Ｎａｔｕｒｅ　３３２：３２３〜３２７、およびＶｅｒｈｏｅｙｅｎら（１９８８）Ｓｃｉｅｎｃｅ　２３９：１５３４〜１５３６）、およびＴ_４ＤＮＡポリメラーゼを使用するギャップオリゴヌクレオチド（ｇａｐｐｅｄ　ｏｌｉｇｏｎｕｃｌｅｏｔｉｄｅ）の酵素充填（ｅｎｚｙｍａｔｉｃ　ｆｉｌｌｉｎｇ−ｉｎ）（Ｑｕｅｅｎら（１９８９）Ｐｒｏｃ．Ｎａｔｌ．Ａｃａｄ．Ｓｃｉ．ＵＳＡ　８６：１００２９〜１００３３）を本発明で使用して、改変されたかもしくは増大された抗原結合能力、および／または減少した免疫原性を有する分子を提供し得る。
【０１０２】
一旦、コード配列が調製または単離されると、このような配列は、任意の適切なベクターまたはレプリコンにクローン化され得る。多数のクローニングベクターが当業者に公知であり、そして適切なクローニングベクターの選択が可能である。適切なベクターとしては、プラスミド、ファージ、トランスポゾン、コスミド、染色体または適切なコントロールエレメントと会合した場合に複製し得るウイルスが挙げられるが、これらに限定されない。
【０１０３】
次いで、コード配列は、発現のために使用される系に依存して、適切なコントロールエレメントの制御下に置かれる。従って、このコード配列は、プロモータ、リボソーム結合部位（細菌の発現のため）、および必要に応じて、オペレーターの制御下に置かれ、その結果、目的のＤＮＡ配列は、適切な形質転換体によってＲＮＡに転写される。このコード配列は、シグナルペプチドまたはリーダー配列（これは後に、翻訳後プロセシングにおいて、宿主によって除去され得る）を含んでも含まなくても良い。例えば、米国特許第４，４３１，７３９号；同第４，４２５，４３７号；同第４，３３８，３９７号を参照のこと。
【０１０４】
制御配列に加えて、宿主細胞の増殖に関連した配列の発現の調節を可能にする調節配列を加えることが所望であり得る。調節配列は、当業者に公知であり、そして例としては、調節化合物を含む、化学的刺激または物理的刺激に対してオンまたはオフにされる遺伝子の発現を引き起こすものが挙げられる。他の型の調節エレメントはまた、ベクター中に存在し得る。例えば、エンハンサーエレメントは、構築物の発現レベルを増加するために本明細書中で使用され得る。例としては、ＳＶ４０初期遺伝子エンハンサー（Ｄｉｊｋｅｍａら（１９８５）ＥＭＢＯ　Ｊ．４：７６１）、ラウス肉腫ウイルスの長末端反復（ＬＴＲ）に由来するエンハンサー／プロモーター（Ｇｏｒｍａｎら（１９８２）Ｐｒｏｃ．Ｎａｔｌ．Ａｃａｄ．Ｓｃｉ．ＵＳＡ　７９：６７７７）およびヒトＣＭＶに由来するエレメント（Ｂｏｓｈａｒｔら（１９８５）Ｃｅｌｌ　４１：５２１）（例えば、ＣＭＶイントロンＡ配列に含まれるエレメント（米国特許第５，６８８，６８８））が挙げられる。発現カセットはさらに、適切な宿主細胞において自律的に複製するための複製起点、１つ以上の選択マーカー、１つ以上の制限部位、高いコピー数についての可能性および強力なプロモーターを含む。
【０１０５】
発現ベクターは、特定のコード配列が適切な制御配列と共にベクター中に位置するように構築され、制御配列に対するコード配列の位置および配向は、コード配列が制御配列の「制御」下で転写されるように存在する（すなわち、制御配列のＤＮＡ分子に結合するＲＮＡポリメラーゼは、コード配列を転写する）。目的の分子をコードする配列の改変は、この目的を達成するために所望である。例えば、いくつかの場合において、適切な配向で配列が制御配列に付着するように（すなわち、リーディングフレームを維持するように）配列を改変することが必要であり得る。制御配列および他の調節配列は、ベクターへの挿入の前にコード配列に連結され得る。あるいは、コード配列は、制御配列および適切な制限部位をすでに含む発現ベクターに直接クローニングされ得る。
【０１０６】
上に説明されるように、目的の抗原の変異体またはアナログを生成することが所望であり得る。これは、特にＮＳ３／４ａに当てはまる。そうするための方法は、例えば、Ｄａｓｍａｈａｐａｔｒａら、米国特許第５，８４３，７５２号およびＺｈａｎｇら、米国特許５，９９０，２７６号において記載されている。目的のアッセイにおける使用のためのこのＨＣＶタンパク質および他のＨＣＶタンパク質の変異体またはアナログは、配列の挿入によって、そして／または配列内での１つ以上のヌクレオチドの置換によって、目的のポリペプチドをコードする配列の一部を欠失することによって調製され得る。ヌクレオチド配列を改変するための方法（例えば、部位特異的変異誘発など）は、当該分野で公知である。例えば、Ｓａｍｂｒｏｏｋら、前出；Ｋｕｎｋｅｌ，Ｔ．Ａ．（１９８５）Ｐｒｏｃ．Ｎａｔｌ．Ａｃａｄ．Ｓｃｉ．ＵＳＡ（１９８５）８２：４４８；Ｇｅｉｓｓｅｌｓｏｄｅｒら（１９８７）ＢｉｏＴｅｃｈｎｉｑｕｅｓ　５：７８６；ＺｏｌｌｅｒおよびＳｍｉｔｈ（１９８３）Ｍｅｔｈｏｄｓ　Ｅｎｚｙｍｏｌ．１００：４６８；Ｄａｌｂｉｅ−ＭｃＦａｒｌａｎｄら（１９８２）Ｐｒｏｃ．Ｎａｔｌ．Ａｃａｄ．Ｓｃｉ　ＵＳＡ　７９：６４０９を参照のこと。
【０１０７】
分子は、昆虫系、哺乳動物系、細菌系、ウイルス系および酵母系を含む広範な種々の系において発現され得、これらは全て当該分野で公知である。
【０１０８】
例えば、昆虫細胞発現系（例えば、バキュロウイルス系）は、当業者に公知であり、そして例えば、ＳｕｍｍｅｒｓおよびＳｍｉｔｈ，Ｔｅｘａｓ　Ａｇｒｉｃｕｌｔｕｒａｌ　Ｅｘｐｅｒｉｍｅｎｔ　Ｓｔａｔｉｏｎ　Ｂｕｌｌｅｔｉｎ　Ｎｏ．１５５５（１９８７）において記載されている。バキュロウイルス／昆虫細胞発現系のための材料および方法は、特に、Ｉｎｖｉｔｏｒｏｇｅｎ，Ｓａｎ　Ｄｉｅｇｏ　ＣＡ（「ＭａｘＢａｃ」キット）から市販されている。同様に、細菌および哺乳動物細胞発現系は、当該分野で周知であり、そして例えば、Ｓａｍｂｒｏｏｋら、前出、において記載されている。酵母発現系はまた、当該分野で公知であり、そして例えば、Ｙｅａｓｔ　Ｇｅｎｅｔｉｃ　Ｅｎｇｉｎｅｅｒｉｎｇ（Ｂａｒｒら編、１９８９）Ｂｕｔｔｅｒｗｏｒｔｈｓ，Ｌｏｎｄｏｎにおいて記載されている。
【０１０９】
上の系を用いる使用のための多数の適切な宿主細胞がまた公知である。例えば、哺乳動物細胞株は、当該分野で公知であり、そして例えば、チャイニーズハムスター卵巣（ＣＨＯ）細胞、ＨｅＬａ細胞、乳児ハムスター腎（ＢＨＫ）細胞、サル腎細胞（ＣＯＳ）、ヒト胚腎細胞、ヒト肝細胞癌細胞（例えば、Ｈｅｐ　Ｇ２）、Ｍａｄｉｎ−Ｄａｒｂｙウシ腎（「ＭＤＢＫ」）細胞、およびその他を含むがこれらに限定されない、Ａｍｅｒｉｃａｎ　Ｔｙｐｅ　Ｃｕｌｔｕｒｅ　Ｃｏｌｌｅｃｔｉｏｎ（ＡＴＣＣ）から入手可能な不死化細胞株を含む。同様に、Ｅ．ｃｏｌｉ、Ｂａｃｉｌｌｕｓ　ｓｕｂｔｉｌｉｓ、およびＳｔｒｅｐｔｏｃｏｃｃｕｓ　ｓｐｐ．のような細菌宿主において、本発明の構築物を用いる用途を見出す。本発明において有用な酵母宿主としては特に、Ｓａｃｃｈａｒｏｍｙｃｅｓ　ｃｅｒｅｖｉｓｉａｅ、Ｃａｎｄｉｄａ　ａｌｂｉｃａｎｓ、Ｃａｎｄｉｄａ　ｍａｌｔｏｓａ、Ｈａｎｓｅｎｕｌａ　ｐｏｌｙｍｏｒｐｈａ、Ｋｌｕｙｖｅｒｏｍｙｃｅｓ　ｆｒａｇｉｌｉｓ、Ｋｌｕｙｖｅｒｏｍｙｃｅｓ　ｌａｃｔｉｓ、Ｐｉｃｈｉａ　ｇｕｉｌｌｅｒｉｍｏｎｄｉｉ、Ｐｉｃｈｉａ　ｐａｓｔｏｒｉｓ、Ｓｃｈｉｚｏｓａｃｃｈａｒｏｍｙｃｅｓ　ｐｏｍｂｅおよびＹａｒｒｏｗｉａ　ｌｉｐｏｌｙｔｉｃａが挙げられる。バキュロウイルス発現ベクターを用いる使用のための昆虫細胞としては特に、Ａｅｄｅｓ　ａｅｇｙｐｔｉ、Ａｕｔｏｇｒａｐｈａ　ｃａｌｉｆｏｒｎｉｃａ、Ｂｏｍｂｙｘ　ｍｏｒｉ、Ｄｒｏｓｏｐｈｉｌａ　ｍｅｌａｎｏｇａｓｔｅｒ、Ｓｐｏｄｏｐｔｅｒａ　ｆｒｕｇｉｐｅｒｄａおよびＴｒｉｃｈｏｐｌｕｓｉａ　ｎｉが挙げられる。
【０１１０】
目的のヌクレオチド配列を含む核酸分子は、当該分野で周知の遺伝子送達技術を使用して、宿主細胞ゲノムに安定に組み込まれ得るか、または適切な宿主細胞中の安定なエピソームエレメント上に維持され得る。例えば、米国特許第５，３９９，３４６号を参照のこと。
【０１１１】
選択される発現系および宿主に依存して、分子は、タンパク質が発現される条件下で、上記の発現ベクターによって形質転換された宿主細胞を増殖することによって生成される。次いで、発現されたタンパク質は、宿主細胞から単離され、そして精製される。発現系が、タンパク質を増殖培地中に分泌する場合、生成物は、培地から直接精製され得る。生成物が分泌されない場合、生成物は細胞溶解物から単離され得る。適切な増殖条件および回収方法の選択は、当該分野の技量内である。
【０１１２】
種々のＨＣＶ抗原の組換え生成が記載されている。例えば、Ｈｏｕｇｈｔｏｎら、米国特許第５，３５０，６７１号；Ｃｈｉｅｎら、Ｊ．Ｇａｓｔｒｏｅｎｔ．Ｈｅｐａｔｏｌ．（１９９３）８：Ｓ３３−３９；Ｃｈｉｅｎら、国際公開番号ＷＯ９３／００３６５；Ｃｈｉｅｎ，Ｄ．Ｙ．，国際公開番号ＷＯ９４／０１７７８を参照のこと。
【０１１３】
（免疫診断アッセイ）
一旦生成されると、上記の抗コア抗体およびＮＳ３／４ａ抗原は、本イムノアッセイにおける使用のために適切な固体支持体上に配置される。本発明の目的のための固体支持体は、不溶性マトリクスである任意の物質であり得、そして剛性または半剛性の表面を有し得る。例示的な固体支持体としては、ニトロセルロースのような基材（例えば、膜またはマイクロタイターウェル形態）；ポリ塩化ビニル（例えば、シートまたはマイクロタイターウェル）；ポリスチレンラテックス（例えば、ビーズまたはマイクロタイタープレート）；ポリフッ化ビニリデン；ジアゾ化紙；ナイロン膜；活性化ビーズ、磁気応答性樹脂などが挙げられるがこれらに限定されない。特定の支持体としては、プレート、ペレット、ディスク、キャピラリー、中空繊維、針、ピン、固体繊維、セルロースビーズ、孔−ガラスビーズ、シリカゲル、ジビニルベンゼンと必要に応じて架橋したポリスチレンビーズ、グラフトされたコポリ（ｃｏ−ｐｏｌｙ）ビーズ、ポリアクリルアミドビーズ、ラテックスビーズ、Ｎ−Ｎ’−ビス−アクリロイルエチレンジアミンと必要に応じて架橋したジメチルアクリルアミドビーズ、および疎水性ポリマーでコーティングしたガラス粒子が挙げられる。
【０１１４】
所望される場合、固体支持体に付加される分子は、スチレン部分またはアクリレート部分を作製するために容易に官能基化され得、従って、ポリスチレン、ポリアクリレート、またはポリイミド、ポリアクリルアミド、ポリエチレン、ポリビニル、ポリジアセチレン、ポリフェニレン−ビニレン、ポリペプチド、多糖類、ポリスルホン、ポリピロール、ポリイミダゾール、ポリチオフェン、ポリエステル、エポキシ、シリカガラス、シリカゲル、シロキサン、ポリホスフェート、ヒドロゲル、アガロース、セルロースなどの他のポリマーへの分子の取り込みを可能にする。
【０１１５】
１つの状況において、固体支持体を、分子が十分に支持体に固定されるような適切な結合条件下で、まずＨＣＶ抗コア抗体およびＮＳ３／４ａエピトープ（本明細書中で集合的に「固相成分」と呼ばれる）、および必要に応じて１つ以上のＭＥＦＡと反応させる。時々、支持体への固定は、より優れた固相結合特性を有するタンパク質への抗原および／または抗体を最初に結合することによって強化され得る。適切なカップリングタンパク質としては、ウシ血清アルブミン（ＢＳＡ）を含む血清アルブミン、キーホールリンペットヘモシニアン、免疫グロブリン分子、チログロブリン、オボアルブミンのような高分子、および当業者に周知の他のタンパク質が挙げられるがこれらに限定されない。支持体に分子を結合させるために使用され得る他の試薬としては、多糖類、ポリ乳酸、ポリグリコール酸、ポリマー性アミノ酸、アミノ酸コポリマーなどが挙げられる。このような分子および抗原にこれらの分子を結合させる方法は、当業者に周知である。例えば、Ｂｒｉｎｋｌｅｙ，Ｍ．Ａ．（１９９２）Ｂｉｏｃｏｎｊｕｇａｔｅ　Ｃｈｅｍ．３：２−１３；Ｈａｓｈｉｄａら（１９８４）Ｊ．Ａｐｐｌ．Ｂｉｏｃｈｅｍ．６：５６−６３；ならびにＡｎｊａｎｅｙｕｌｕおよびＳｔａｒｏｓ（１９８７）Ｉｎｔｅｒｎａｔｉｏｎａｌ　Ｊ．ｏｆ　Ｐｅｐｔｉｄｅ　ａｎｄ　Ｐｒｏｔｅｉｎ　Ｒｅｓ．３０：１１７−１２４を参照のこと。
【０１１６】
固体支持体を固相成分と反応させた後、任意の非固定固相成分を、洗浄することによって支持体から取り除き、次いで支持体結合成分を、適切な結合条件下で、ＨＣＶ抗体および抗原（本明細書中で集合的に「リガンド分子」と呼ばれる）を含むことが疑われている生物学的サンプルと接触させる。任意の結合していないリガンド分子を除去するために洗浄した後、支持体に結合した抗コア抗体と異なるエピトープに対して指向する第２の抗コア抗体を、適切な結合条件下で添加する。添加される抗コア抗体は、上記のように、検出可能な標識を含み、そして支持体に結合した抗コア抗体と反応したサンプル中に存在し得る任意のコア抗原を結合するように作用する。ＮＳ３／４Ａエピトープと順番に反応したサンプル中に存在する抗体と反応し得る１つ以上の抗原もまた、添加される。上記で説明したように、抗原は、代表的に、ＨＣＶポリタンパク質のＮＳ３領域から誘導され、特に、ＨＣＶのｃ３３ｃ領域から誘導される。これらから誘導されるエピトープの領域の説明に関しては、Ｈｏｕｇｈｔｏｎら、米国特許第５，３５０，６７１号；Ｃｈｉｅｎら、Ｐｒｏｃ．Ｎａｔｌ．Ａｃａｄ．Ｓｃｉ（１９８９）８９：１００１１−１００１５；国際公開番号ＷＯ９３／００３６５；ならびに、一般に所有された、許可された米国特許出願第０８／４０３，５９０および０８／４４４，８１８を参照のこと。この抗原に対する標識抗体もまた、添加される。従って、抗体は、サンプル中に存在する抗ＮＳ３抗体と反応した抗原に結合する。この目的のために、ｃ３３ｃエピトープは、ｃ３３ｃとヒトスーパーオキシドジスムターゼ（ｈＳＯＤ）との間の融合として好都合に提供され、例えば、Ｈｏｕｇｈｔｏｎら、米国特許第５，３５０，６７１号に記載される方法によって組換え産生され得る。ヒトＳＯＤのヌクレオチド配列およびアミノ酸配列は、公知であり、Ｈａｌｌｅｗｅｌｌら、米国特許第５，７１０，０３３号に報告される。従って、ヒトＳＯＤに対する標識抗体は、ＮＳ３／４ａエピトープ、このエピトープと反応するサンプル中の任意の抗体、およびサンプル中のその抗体と順に結合するポリペプチドの間で形成される複合体の存在を検出するために使用され得る。
【０１１７】
ＭＥＦＡが固体支持体上に存在する場合、ＭＥＦＡ上に存在する抗原に結合する生物学的サンプル由来の抗体と反応性である１つ以上のさらなる抗原もまた、アッセイに添加され得る。この状況において特に有用なことは、ＨＣＶのコア領域由来の抗原であり、より詳細には、ＨＣＶポリタンパク質の１１９Ｎ末端コアアミノ酸を含むｃ２２抗原由来であることである。ｃ２２由来の１つの特定の抗原は、ポリタンパク質のアミノ酸Ｌｙｓ_１０〜Ｓｅｒ_９９ならびに通常存在するＡｒｇ４７の欠失および４４位にＴｒｐの代わりのＬｅｕを含む、ｃ２２ｋｓ　Δ４７−Ｌ４４Ｗである。上記のｃ３３ｃエピトープに関して、この抗原は、ｈＳＯＤおよびヒトＳＯＤに対するその標識抗体との融合として提供され、サンプル中に存在する抗体と、ＮＳ３／４ａエピトープおよび／またはＭＥＦＡとの間に形成される複合体（これらの複合体はまた、ＨＣＶ抗原（例えば、ｃ３３ｃおよびｃ２２）と結合する）の存在を検出するために使用され得る。
【０１１８】
より詳細には、ＥＬＩＳＡ方法を、使用し得、ここで、マイクロタイタープレートのウェルを、固相成分と接触させる。次いで、リガンド分子を含むかまたは含むことが疑われる生物学的サンプルを、コーティングされたウェルに添加する。リガンド−分子の固定化された固相成分への結合を可能にするのに十分なインキュベーション期間の後、プレートを洗浄して、未結合の部分を除去し得、そして、検出可能に標識された２次結合分子（標識抗コア抗体）、ＮＳ３エピトープ含有分子、およびＮＳ３エピトープ含有部分に対する抗体を、添加される。これらの分子を、任意の捕獲サンプル抗原および抗体と反応させ、プレートを洗浄し、そして標識抗体の存在が、当該分野で周知の方法を用いて検出される。
【０１１９】
上記のアッセイ試薬（抗体および抗原を有するイムノアッセイ固体支持体、ならびに捕獲サンプルと反応する抗体および抗原を含む）をまた、適切な指示書および他の必要な試薬と共にキット中に提供して、上記のようにイムノアッセイを実行し得る。このキットはまた、使用する特定のイムノアッセイに依存して、適切な標識ならびに他のパッケージングされた試薬および材料（すなわち、洗浄緩衝液など）を含み得る。標準的なイムノアッセイ（例えば、上記に記載されるもの）を、これらのキットを使用して実行し得る。
【０１２０】
（ＩＩＩ．実験）
以下は、本発明を実行するための特定の実施形態の例である。本実施例は、例示目的のみのために提供され、そして本発明の範囲をいずれにも限定することは意図されない。
【０１２１】
使用した数値（例えば、量、温度など）に関して精度を確実にするための努力がなされたが、いくらかの実験誤差および偏差は、当然許容されるべきである。
【０１２２】
（実施例１）
（ＨＣＶ抗原／抗体の組み合わせイムノアッセイ）
本発明のＨＣＶ抗原／抗体の組み合わせイムノアッセイを、セロコンバージョン検出限界を試験し、そしてこれらの限界を以下のような他の市販で入手されるアッセイに対する限界と比較するために、他のＨＣＶアッセイと比較した。
【０１２３】
（Ａ．材料および方法）
（血液サンプル）市販のヒト血液サンプルのパネルを使用した。このようなパネルは、例えば、Ｂｏｓｔｏｎ　Ｂｉｏｍｅｄｉｃａ，Ｉｎｃ．，Ｗｅｓｔ　Ｂｒｉｄｇｅｗａｔｅｒ，ＭＡ（ＢＢＩ）；Ｂｉｏｃｌｉｎｉｃａｌ　Ｐａｒｔｎｅｒｓ，Ｆｒａｎｋｌｉｎ，ＭＡ（ＢＣＰ）；およびＮｏｒｔｈ　Ａｍｅｒｉｃａｎ　Ｂｉｏｌｏｇｉｃｓ，Ｉｎｃ．，ＢｏｃｏＲａｔａｎ，ＦＬ（ＮＡＢＩ）から入手可能である。表５および６に示した日は、血液を被験体から収集した日である。
【０１２４】
（モノクローナル抗体）モノクローナル抗体ｃ１１−３、ｃ１１−７およびｃ１１−１４を、Ｏｒｔｈｏ　Ｃｌｉｎｉｃａｌ　Ｄｉａｇｎｏｓｔｉｃｓ，Ｒａｒｉｔａｎ，Ｎｅｗ　Ｊｅｒｓｅｙから入手した。ｃ１１−３およびｃ１１−７抗体は、コアのＮ末端部分（ＨＣＶ１ポリペプチドに対する番号付けでアミノ酸１０〜５３）に対して指向する。ｃ１１−１４モノクローナル抗体は、コアのＣ末端部分（ＨＣＶ１ポリペプチドに対する番号付けでアミノ酸１２０〜１３０）に対して指向する。ｃ１１−１４抗体は、標準的な手順を用いて西洋ワサビペルオキシダーゼ（ＨＲＰ）に結合体化されていた。
【０１２５】
モノクローナル抗体５Ａ−３は、ＳＯＤのアミノ酸１〜６５に対して指向された抗ＳＯＤ抗体であり、標準的な技術を使用して作製された。この抗体は、上記のようにＨＲＰに結合体化されていた。
【０１２６】
（Ｂ．抗原）
ｃ３３ｃ抗原（２６６アミノ酸、ＨＣＶ１ポリタンパク質のアミノ酸１１９２〜１４５７）を、５−１−１抗原の合成について記載された方法（Ｃｈｏｏら、Ｓｃｉｅｎｃｅ（１９８９）２４４：３５９−３６２）によって、Ｅ．ｃｏｌｉ中で内部ＳＯＤ融合ポリペプチドとして発現させた。組換え抗原を、Ｃｈｉｅｎら、Ｐｒｏｃ．Ｎａｔｌ．Ａｃａｄ．Ｓｃｉ．（１９８９）８９：１００１１−１００１５に記載されるように精製した。ＳＯＤ−ｃ３３ｃの生産手順については、Ｈｏｕｇｈｔｏｎら、米国特許第５，３５０，６７１号もまた、参照のこと。
【０１２７】
本アッセイに使用されるＮＳ３／４ａエピトープは、図３に特定される配列を有するコンホメーションエピトープである。
【０１２８】
（Ｃ．イムノアッセイ形式）
Ａｂｂｏｔｔ　ＰＲＩＳＭアッセイ（Ａｂｂｏｔｔ　Ｌａｂｏｒａｔｏｒｉｅｓ，Ａｂｂｏｔｔ　Ｐａｒｋ，ＩＬ）は、市販され、そして抗体に基づく検出アッセイである。このアッセイを、製造者らの指示書を用いて実行した。
【０１２９】
ＯＲＴＨＯ　ＨＣＶ　バージョン　３．０　ＥＬＩＳＡ試験システム（本明細書中、Ｏｒｔｈｏ　３．０アッセイと称する、Ｏｒｔｈｏ　Ｃｌｉｎｉｃａｌ　Ｄｉａｇｎｏｓｔｉｃｓ，Ｒａｒｉｔａｎ，Ｎｅｗ　Ｊｅｒｓｅｙ）は、抗体に基づく検出アッセイである。このアッセイを、製造者らの指示書を用いて実行した。
【０１３０】
Ｒｏｃｈｅ　Ａｍｐｌｉｃｏｒアッセイ（Ｒｏｃｈｅ，Ｐｌｅａｓａｎｔ，ＣＡ）は、市販のＰＣＲに基づくアッセイである。このアッセイを、製造者らの指示書を用いて実行した。
【０１３１】
Ｇｅｎ−Ｐｒｏｂｅ　ＴＭＡアッセイ（Ｓａｎ　Ｄｉｅｇｏ，ＣＡ）は、市販の転写媒介増幅アッセイである。このアッセイを、製造者らの指示書を用いて実行した。
【０１３２】
Ｏｒｔｈｏ抗原アッセイ（Ｏｒｔｈｏ　Ｃｌｉｎｉｃａｌ　Ｄｉａｇｎｏｓｔｉｃｓ，Ｒａｒｉｔａｎ，Ｎｅｗ　Ｊｅｒｓｅｙ）は、抗原に基づく検出アッセイである。このアッセイを、製造者らの指示書を用いて実行した。
【０１３３】
本ＨＣＶ抗原／抗体の組み合わせイムノアッセイを、以下のように実行した。１×リン酸緩衝化生理食塩水（ＰＢＳ）（ｐＨ７．４）中、各々４ｍｇ／ｍＬの精製モノクローナル抗体Ｃ１１−７とＣ１１−３とを合わせ、そしてウェルを混合した。９０ｎｇのＮＳ３／４ａ組換え抗原を、同じコーティング緩衝液に添加した。この溶液は、コーティング前に３０分間混合した。２００ｍＬの上記の溶液を、９６ウェル　Ｃｏｓｔａｒ培地結合マイクロタイタープレート（Ｃｏｒｉｎｇ，Ｉｎｃ．）へ各ウェルにつき添加した。プレートを、１５〜３０℃で１６〜２４時間インキュベートした。プレートを、ｄＨ_２Ｏを用いて２回洗浄し、続いて、３００μＬ／ウェル　後コーティング緩衝液（１％　ウシ血清アルブミン（ＢＳＡ），１×ＰＢＳ）を用いて１時間洗浄し、そして３００μＬ／ウェルの安定緩衝液（１×ＰＢＳ，１％　ＢＳＡ，マンニトール、ポリエチレングリコール（ＰＥＧ），ゼラチン）を用いて１時間洗浄した。プレートを吸引し、そして、凍結乾燥機中、４℃で２４時間乾燥させた。プレートを、乾燥剤と一緒に小袋に入れた。
【０１３４】
抗原／抗体の組み合わせイムノアッセイを実行するために、１００μＬの増強溶解緩衝液（１％　Ｎ−ラウリルサルコシン、０．６５Ｍ　ＮａＣｌ、５０ｍｇ／ｍＬ　マウスＩｇＧ試験等級（Ｓｉｇｍａ，Ｓｔ．Ｌｏｕｉｓ，ＭＯ）、１％　スルフヒドリル改変ＢＳＡ（Ｂａｙｅｒ）、０．１％　カゼイン）を、このプレートに添加した。次いで、１００ｍＬのサンプルを、添加した。これを、振盪器上、４０℃で１時間インキュベートした。プレートを、Ｏｒｔｈｏ　Ｐｌａｔｅ　Ｗａｓｈｅｒ上、１×ＰＢＳ、０．１％　Ｔｗｅｅｎ−２０を用いて６回洗浄した。２００ｍＬの結合溶液（２５０ｎｇ／アッセイ　ＳＯＤ−ｃ３３ｃ抗原を有する１：７５希釈ｃ１１−１４−ＨＲＰ、ならびにＳＯＤ抽出物を含まないＨＣＶ３．０サンプル希釈物中の１：５０００希釈マウス抗ＳＯＤ−ＨＲＰ（ＯＲＴＨＯ　ＨＣＶ　バージョン　３．０　ＥＬＩＳＡ試験システム（Ｏｒｔｈｏ　Ｃｌｉｎｉｃａｌ　Ｄｉａｇｎｏｓｔｉｃｓ，Ｒａｒｉｔａｎ，Ｎｅｗ　Ｊｅｒｓｅｙ）（全て、添加の３０分前に調製した）。この溶液を、振盪しながら４０℃で４５分間インキュベートした。これを、上記のように６回洗浄し、そして２００ｍＬの基質溶液（１ＯＰＤ錠剤／１０ｍＬ）を、添加した。ＯＰＤ錠剤は、西洋ワサビペルオキシダーゼ反応の色素発生のためのｏ−フェニレンジアミンジハイドロクロライドおよび過酸化水素を含み、そしてＳｉｇｍａ，Ｓｔ．Ｌｏｕｉｓ，ＭＯから市販されている。これを、３０分間、１５〜３０℃で暗闇中インキュベートした。この反応を、５０ｍＬ　４Ｎ　Ｈ_２ＳＯ_４の添加によって停止させ、そしてプレートを、コントロールとしての６９０ｎｍでの吸光度に対して、４９２ｎｍで読み取った。
【０１３５】
（Ｄ．結果）
種々のアッセイの結果を、表５および表６に示し、これは示したようにＨＣＶ感染に曝露された血液サンプルで実施した２つの別々の実験を示す。斜線領域は、ウイルスの検出を示す。以下に示されるように、Ｃｈｉｒｏｎの組み合わせ抗原／抗体アッセイは、全てのサンプル中でセロコンバージョンを検出し、一方全ての他の抗体および抗原ベースのアッセイは、少なくとも１つのサンプルにおいてセロコンバージョンを検出することに失敗した。特に、いずれの抗体ベースのアッセイも、少なくとも１８日目までセロコンバージョンを検出しなかった（表５）。表６は、いずれの抗体ベースのアッセイも、ＨＣＶ感染の存在を２２日目で検出しなかったことを示す。さらに、Ｏｒｔｈｏ抗原ベースのアッセイは、８５日目からセロコンバージョンの検出に失敗した。
【０１３６】
従って、上記の結果に基づいて、新規の組み合わせ抗体／抗原アッセイが、他の従来の抗体および抗原ベースのアッセイを用いて得られる偽陰性数を減少させることは明らかである。
【０１３７】
【表５】

【０１３８】
【表６】

（実施例２）
（ＴｈｒからＰｒｏおよびＳｅｒからＩｌｅへの置換を伴うＮＳ３／４ａコンホメーションエピトープの産生）
ＮＳ３／４ａのコンホメーションエピトープを以下の通りに得た。このエピトープは、図４Ａ〜４Ｄで指定した配列を有し、ネイティブな配列とは４０３位（ＨＣＶ−１全長配列のアミノ酸１４２８）および４０４位（ＨＣＶ−１全長配列のアミノ酸１４２９）で異なる。特に、ネイティブな配列の１４２８位で通常生じるＴｈｒはＰｒｏへと変異しており、そしてネイティブな配列の１４２９位で生じるＳｅｒは、Ｉｌｅへと変異している。
【０１３９】
特に、用いられる酵母発現ベクターは、上記のｐＢＳ２４．１であった。このイムノアッセイにおいて用いられる代表的なＮＳ３／４ａエピトープをコードするプラスミドｐｄ．ｈｃｖ１ａ．ｎｓ３ｎｓ４ａＰＩを、以下の通りに作製した。二工程手順を用いた。最初に、以下のＤＮＡ片を一緒に連結した：（ａ）５’ＨｉｎｄＩＩＩクローニング部位、続いて配列ＡＣＡＡＡＡＣＡＡＡ、イニシエーターＡＴＧ、およびアミノ酸１０２７で始まりアミノ酸１０４６のＢｇｌＩ部位へと続くＨＣＶ１ａについてのコドンを提供する合成オリゴヌクレオチド；（ｂ）ｐＡｃＨＬＴｎｓ３ｎｓ４ａＰＩ由来の６８３ｂｐのＢｇｌＩ−ＣｌａＩ制限フラグメント（アミノ酸１０４６〜１２７４をコードする）；ならびに（ｃ）ＨｉｎｄＩＩＩおよびＣｌａＩで消化し、脱リン酸化し、そしてゲル精製したｐＳＰ７２ベクター（Ｐｒｏｍｅｇａ，Ｍａｄｉｓｏｎ，ＷＩ，ＧｅｎＢａｎｋ／ＥＭＢＬ登録番号Ｘ６５３３２）。プラスミドｐＡｃＨＬＴｎｓ３ｎｓ４ａＰＩを、ＢＤ　Ｐｈａｒｍｉｎｇｅｎ（Ｓａｎ　Ｄｉｅｇｏ，ＣＡ）から市販されているバキュロウイルス発現ベクターであるｐＡｃＨＬＴから誘導した。特に、ｐＡｃＨＬＴ　ＥｃｏＲＩ−ＰｓｔＩベクターならびに以下のフラグメントを調製した：ＨＣＶ−１ゲノムのアミノ酸１０２７〜１３３６に対応する９３５ｂｐのＥｃｏＲＩ−ＡｌｗｎＩ；ＨＣＶ−１ゲノムのアミノ酸１３３６〜１４１９に対応する２４７ｂｐのＡｌｗｎＩ−ＳａｃＩＩ；ＨＣＶ−１ゲノムのアミノ酸１４４９〜１５０９に対応する１７５ｂｐのＨｉｎｆＩ−ＢｇｌＩ；ＨＣＶ−１ゲノムのアミノ酸１５１０〜１７１１に対応する６１９ｂｐのＢｇｌＩ−ＰｓｔＩ＋転写終結コドン。ＨＣＶ−１ゲノムのアミノ酸１４２０〜１４４８に対応し、ＰＩ変異（Ｐｒｏへと変異させたＴｈｒ−１４２８、Ｉｌｅへと変異させたＳｅｒ−１４２９）を含む、合成によって作製した９１ｂｐのＳａｃＩＩ−ＨｉｎｆＩフラグメントを上記の１７５ｂｐのＨｉｎｆＩ−ＢｇｌＩフラグメントおよび６１９ｂｐのＢｇｌＩ−ＰｓｔＩフラグメントと連結し、そしてＳａｃＩＩおよびＰｓｔＩで消化したｐＧＥＭ−５Ｚｆ（＋）ベクター中にサブクローン化した。ｐＧＥＭ−５Ｚｆ（＋）は、市販のＥ．ｃｏｌｉベクター（Ｐｒｏｍｅｇａ，Ｍａｄｉｓｏｎ，ＷＩ，ＧｅｎＢａｎｋ／ＥＭＢＬ登録番号Ｘ６５３０８）である。コンピテントなＨＢ　１０１細胞の形質転換、個々のクローンのミニスクリーニング分析および配列確認後、ｐＧＥＭ５．ＰＩクローン２由来の８８５ｂｐのＳａｃＩＩ−ＰｓｔＩフラグメントをゲル精製した。このフラグメントを、上記のＥｃｏＲＩ−ＡｌｗｎＩ　９３５ｂｐフラグメント、ＡｌｗｎＩ−ＳａｃＩＩ　２４７ｂｐフラグメントおよびｐＡｃＨＬＴ　ＥｃｏＲＩ−ＰｓｔＩベクターと連結した。得られた構築物を、ｐＡｃＨＬＴｎｓ３ｎｓ４ａＰＩと命名した。
【０１４０】
上記の連結混合物をＨＢ１０１コンピテント細胞中に形質転換し、そして１００μｇ／ｍｌアンピシリンを含有するＬｕｒｉａ寒天プレートにプレーティングした。個々のクローンのミニプレップ分析によって推定のポジティブを同定し、そのうちの２つを増幅した。ｐＳＰ７２　１ａＨＣのクローン番号１およびクローン番号２についてのプラスミドＤＮＡを、Ｑｉａｇｅｎ　Ｍａｘｉｐｒｅｐキットを用いて調製し、そして配列決定した。
【０１４１】
次いで、以下のフラグメントを一緒に連結した：（ａ）ｐＳＰ７２１ａＨＣ　＃１由来の７６１ｂｐのＨｉｎｄＩＩＩ−ＣｌａＩフラグメント（ｐＳＰ７２．１ａＨＣを、以下を一緒に連結することによって作製した：ＨｉｎｄＩＩＩおよびＣｌａＩで消化したｐＳＰ７２、５’ＨｉｎｄＩＩＩクローニング部位を提供する合成オリゴヌクレオチド、続いて配列ＡＣＡＡＡＡＣＡＡＡ、開始コドンＡＴＧおよびアミノ酸１０２７で始まり、アミノ酸１０４６でのＢｇｌＩＩ部位へと続く、ＨＣＶ１ａについてのコドン、ならびにｐＡｃＨＬＴｎｓ３ｎｓ４ａＰＩ由来の６８３ｂｐのＢｇｌＩＩ−ＣｌａＩ制限フラグメント（アミノ酸１０４６〜１２７４をコードする））；（ｂ）酵母ハイブリッドプロモーターＡＤＨ２／ＧＡＰＤＨについての１３５３ｂｐのＢａｍＨＩ−ＨｉｎｄＩＩＩフラグメント；（ｃ）ｐＡｃＨＬＴｎｓ３ｎｓ４ａＰＩ由来の１３２０ｂｐのＣｌａＩ−ＳａｌＩフラグメント（Ｔｈｒ　１４２８がＰｒｏへと変異し、そしてＳｅｒ　１４２９がＩｌｅへと変異したＨＣＶ１ａアミノ酸１０４６〜１７１１をコードする）；ならびに（ｄ）ＢａｍＨＩおよびＳａｌＩで消化し、脱リン酸化し、そしてゲル精製したｐＢＳ２４．１酵母発現ベクター。連結混合物をコンピテントなＨＢ１０１中へと形質転換し、そして１００μｇ／ｍｌアンピシリンを含有するＬｕｒｉａ寒天プレートにプレーティングした。個々のコロニーのミニプレップ分析によって、予想された３４４６ｂｐのＢａｍＨＩ−ＳａｌＩ挿入物（ＡＤＨ２／ＧＡＰＤＨプロモーター、イニシエーターコドンＡＴＧおよびアミノ酸１０２７〜１７１１のＨＣＶ１ａ　ＮＳ３／４ａ（図４Ａ〜図４Ｄのアミノ酸１〜６８６として示す）（Ｔｈｒ　１４２８（図４Ａ〜４Ｄのアミノ酸位置４０３）がＰｒｏへと変異し、そしてＳｅｒ　１４２９（図４Ａ〜４Ｄのアミノ酸位置４０４）がＩｌｅへと変異している）から構成される）を有するクローンを同定した。この構築物を、ｐｄ．ＨＣＶ１ａ．ｎｓ３ｎｓ４ａＰＩと命名した（図５を参照のこと）。
【０１４２】
Ｓ．ｃｅｒｅｖｉｓｉａｅ株ＡＤ３をｐｄ．ＨＣＶ１ａ．ｎｓ３ｎｓ４ａＰＩで形質転換し、そして１つの形質転換体を、培地中のグルコースの枯渇後の発現についてチェックした。組換えタンパク質は、クマシーブルー染色によって検出し、そしてＮＳ３のヘリカーゼドメインに対するポリクローナル抗体を用いて免疫ブロット分析によって確認したところ、酵母において高いレベルで発現された。
【０１４３】
（実施例３）
（ＮＳ３／４ａコンホメーションエピトープの精製）
ＮＳ３／４ａコンホメーションエピトープを以下の通りに精製した。ＮＳ３／４ａエピトープを発現する、上記由来のＳ．ｃｅｒｅｖｉｓｉａｅ細胞を、上記の通りに収集した。この細胞を溶解緩衝液（５０ｍＭ　Ｔｒｉｓ（ｐＨ８．０）、１５０ｍＭ　ＮａＣｌ、１ｍＭ　ＥＤＴＡ、１ｍＭ　ＰＭＳＦ、０．１μＭペプスタチン、１μＭロイペプチン）中に懸濁し、そして１：１：１の細胞：緩衝液：０．５ｍｍガラスビーズの比でガラスビーズを用いて、Ｄｙｎｏ−Ｍｉｌｌ（Ｗａｂ　Ｗｉｌｌｙ　Ａ．Ｂａｃｈｏｆｏｎ，Ｂａｓｅｌ，Ｓｗｉｔｚｅｒｌａｎｄ）またはそれに相当する装置において溶解した。溶解産物を４℃で３０１００×ｇで３０分間遠心分離し、そして不溶タンパク質画分を含むペレットを洗浄緩衝液（６ｍｌ／ｇ出発細胞ペレット重量）に添加し、そして室温で１５分間揺り動かした。洗浄緩衝液は、５０ｍＭ　ＮａＰＯ_４（ｐＨ８．０）、０．３Ｍ　ＮａＣｌ、５ｍＭ　β−メルカプトエタノール、１０％グリセロール、０．０５％オクチルグルコシド、１ｍＭ　ＥＤＴＡ、１ｍＭ　ＰＭＳＦ、０．１μＭペプスタチン、１μＭロイペプチンからなっていた。細胞破片を、３０１００×ｇで３０分間、４℃での遠心分離によって取り出した。上清を捨て、そしてペレットを保持した。
【０１４４】
タンパク質を、このペレットから以下の通りに抽出した。６ｍｌ／ｇの抽出緩衝液を添加し、そして室温で１５分間揺り動かした。この抽出緩衝液は、５０ｍＭ　Ｔｒｉｓ（ｐＨ８．０）、１Ｍ　ＮａＣｌ、５ｍＭ　β−メルカプトエタノール、１０％グリセロール、１ｍＭ　ＥＤＴＡ、１ｍＭ　ＰＭＳＦ、０．１μＭペプスタチン、１μＭロイペプチンからなっていた。これを、３０１００×ｇで３０分間、４℃で遠心分離した。上清を保持し、そして以下の式を用いて１７．５％になるように硫酸アンモニウムを添加した：上清の容積（ｍｌ）×ｘ％硫酸アンモニウム／（１−ｘ％硫酸アンモニウム）＝上清へと添加される４．１Ｍ飽和硫酸アンモニウムのｍｌ。氷上で攪拌しながら硫酸アンモニウムを滴下し、そして溶液を氷上で１０分間攪拌した。溶液を１７７００×ｇで３０分間、４℃で遠心分離し、そしてペレットを保持し、そして２℃〜８℃で４８時間まで保存した。
【０１４５】
ペレットを再懸濁し、そしてポリＵカラム（Ｐｏｌｙ　Ｕ　Ｓｅｐｈａｒｏｓｅ　４Ｂ，Ａｍｅｒｓｈａｍ　Ｐｈａｒｍａｃｉａ）に４℃で以下の通りに流した。ペレットを、ペレットの重量１グラムあたり６ｍｌのポリＵ平衡化緩衝液中に再懸濁した。この平衡化緩衝液は、２５ｍＭ　ＨＥＰＥＳ（ｐＨ８．０）、２００ｍＭ　ＮａＣｌ、５ｍＭ　ＤＴＴ（新たに添加した）、１０％グリセロール、１．２オクチルグルコシドからなっていた。この溶液を４℃で１５分間で揺り動かし、そして３１０００×ｇで３０分間、４℃で遠心分離した。
【０１４６】
ポリＵカラム（１グラムの出発ペレット重量あたり１ｍｌ樹脂）を調製した。線形流速は６０ｃｍ／時間であり、そしてパッキング流速は６０ｃｍ／時間の１３３％であった。このカラムを平衡化緩衝液で平衡化し、そして再懸濁した硫酸アンモニウムペレットの上清を、この平衡化したカラムにローディングした。このカラムを、平衡化緩衝液でベースラインになるまで洗浄し、そしてタンパク質を、以下のポリＵ溶出緩衝液中で１段階溶出で溶出させた：２５ｍＭ　ＨＥＰＥＳ（ｐＨ８．０）、１Ｍ　ＮａＣｌ、５ｍＭ　ＤＴＴ（新たに添加した）、１０％グリセロール、１．２オクチルグルコシド。カラムの溶出物をＳＤＳ−ＰＡＧＥ（クマシー染色した）で泳動し、そしてアリコートを凍結して−８０℃で保存した。ＮＳ３／４ａエピトープの存在を、ＮＳ３プロテアーゼドメインに対するポリクローナル抗体および５−１−１エピトープ（ＨＣＶ　４ａ）に対するモノクローナル抗体を用いたウェスタンブロットによって確認した。
【０１４７】
さらに、プロテアーゼ酵素活性を、以下の通りにして精製の間、モニタリングした。ＮＳ４Ａペプチド（ＫＫＧＳＶＶＩＶＧＲＩＶＬＳＧＫＰＡＩＩＰＫＫ）およびＮＳ３／４ａコンホメーションエピトープ含有サンプルを９０μｌの反応緩衝液（２５ｍＭ　Ｔｒｉｓ（ｐＨ７．５）、０．１５Ｍ　ＮａＣｌ、０．５ｍＭ　ＥＤＴＡ、１０％グリセロール、０．０５　ｎ−ドデシルＢ−Ｄ−マルトシド、５ｍＭ　ＤＴＴ）中に希釈し、そして室温で３０分間混合させた。９０μｌの混合物をマイクロタイタープレート（Ｃｏｓｔａｒ，Ｉｎｃ．，Ｃｏｒｎｉｎｇ，ＮＹ）に添加し、そして１０μｌのＨＣＶ基質（ＡｎａＳｐｅｃ，Ｉｎｃ．，Ｓａｎ　Ｊｏｓｅ　ＣＡ）を添加した。プレートを混合し、そしてＦｌｕｏｓｔａｒプレートリーダーで読み取った。結果を、１分間あたりの相対蛍光単位（ＲＦＵ）で表した。
【０１４８】
これらの方法を用いて、１Ｍ　ＮａＣｌ抽出物の産物は３．７ＲＦＵ／分の活性を含んでおり、硫酸アンモニウム沈澱物は７．５ＲＦＵ／分の活性を有し、そしてポリＵ精製の産物は１８．５ＲＦＵ／分の活性を有していた。
【０１４９】
（実施例４）
（競合研究）
以下の競合研究を、ＮＳ３／４ａコンホメーションエピトープが、他のＨＣＶ抗原と異なる抗体を検出するか否かを評価するために実施した。詳細には、ＮＳ３／４ａ抗原を、以下のようにしてｃ２００抗原と比較した。
【０１５０】
上記のように生成された０．５μｇおよび１．０μｇのＮＳ３／４ａまたはｃ２００（Ｈｅｐａｔｏｌｏｇｙ（１９９２）１５：１９−２５、ＯＲＴＨＯ　ＨＣＶ　Ｖｅｒｓｉｏｎ　３．０　ＥＬＩＳＡ　Ｔｅｓｔ　Ｓｙｓｔｅｍ，Ｏｒｔｈｏ−Ｃｌｉｎｉｃａｌ　Ｄｉａｇｎｏｓｔｉｃｓ，Ｒａｒｉｔａｎ，Ｎｅｗ　Ｊｅｒｓｅｙにおいて利用可能）を、総量２２０μｌ（１×ＰＢＳ）で２０μｌのサンプルＰＨＶ９１４−５（感染個体の血液から獲得された初期セロコンバージョンの採血）と混合した。この混合物を、マイクロウェル中で３７℃で１時間インキュベートした。次いでこの混合物を、ＮＳ３／４ａコーティングしたプレートに移し、そして３７℃で１時間インキュベートした。プレートを洗浄し、そして以下のようにしてアッセイした。
【０１５１】
１μｇのｃ２００抗原を、総量約２２０μｌ中の１０μｌのサンプルＰＨＶ９１４−５に添加した。この混合物を、マイクロウェル中で３７℃で１時間インキュベートし、そして２００μｌをＮＳ３／４ａコーティングしたプレートに移し（１００ｎｇ／アッセイ）、そして３７℃で１時間インキュベートした。プレートを、１×ＰＢＳ、０．１％　Ｔｗｅｅｎ−２０を用いて５回洗浄した。２００μｌの結合溶液（上記）を添加し、そしてプレートをインキュベートし、そしてアッセイした。ＰＨＶ９１４−５および１×ＰＢＳからなるコントロール（抗原を含まない）もまた上記の通り処理した。
【０１５２】
結果を表７に示す。４列目に示される％阻害結果は、３列目−｛（２列目／３列目）×１００｝として算出する。見られ得る通り、これらのデータは、ＮＳ３４ａが、初期セロコンバージョン抗体により中和され、そしてｃ２００は中和されないことを示す。ＰＨＶ９１４−５　ｃ３３ｃ初期セロコンバージョンパネルのメンバーにおける抗体が、プレート上にコーティングされたＮＳ３４ａと反応する場合、強いシグナルを達成した。ｃ２００抗原は、これらの抗体により中和されなかった。このことは、表７の上パネルに示される。ＮＳ３４ａをＰＨＶ９１４−５サンプルと混合した場合、それは中和され、従って、マイクロプレート上にコーティングされたＮＳ３４ａと反応する抗体は、サンプル中に存在しなかった。これらのデータは、ＮＳ３４ａが、ｃ２００により検出される抗体とは異なるクラスの抗体を検出し得ることを示す。
【０１５３】
【表７】

（実施例５）
（ＮＳ３／４ａコンホメーションエピトープの安定性の研究）
アッセイの性能に対するＮＳ３／４ａエピトープの安定性の役割を評価するために、以下の研究を実施して、室温での時間に対するＮＳ３／４ａの免疫反応性を決定した。小アリコートのストックＮＳ３／４ａを、表８に示される間隔で、室温に静置し、次いで凍結させた。全てのバイアルを同時にコーティングし、そして２つの初期ＮＳ３セロコンバージョンパネルに対して試験した。
【０１５４】
表８に見られる通り、ＮＳ３／４ａストックは安定ではなく、そして時間と共に免疫反応性が減少する。さらに、ＮＳ３／４ａのコンホメーションを維持することは、免疫反応性のために必須である。
【０１５５】
さらなる安定性の研究を以下の通り実施した。標準的な手順を用いてＮＳ３／４ａに対して作製された２つのコンホメーションモノクローナル抗体を、抗ＨＣＶ初期セロコンバージョンパネルと置き換えた。ストックＮＳ３／４ａバイアルを、室温にて３、６、および２４時間間隔で保存した。凍結バイアル由来のＮＳ３／４ａを９０ｎｇ／ｍｌでコーティングし、そして上記の手順を用いてアッセイした。結果は、２つのモノクローナル抗体が実際にコンホメーション抗体であり、そしてそれらの反応性は室温でのストックＮＳ３／４ａ抗原の操作に対して感受性であったことを示唆した。ポジティブコントロールのモノクローナル抗体の反応性は変化しなかった。
【０１５６】
【表８】

（実施例６）
（変性ＮＳ３／４ａに対するＮＳ３／４ａコンホメーションエピトープの免疫反応性）
上記のように生成されたＮＳ３／４ａコンホメーションエピトープの免疫反応性を、ＮＳ３／４ａコンホメーションエピトープ調製物に最終濃度２％でＳＤＳを添加することにより変性させたＮＳ３／４ａと比較した。変性ＮＳ３／４ａおよび立体構造性のＮＳ３／４ａを、上記のようにマイクロタイタープレート上にコーティングした。ｃ２００抗原（Ｈｅｐａｔｏｌｏｇｙ（１９９２）１５：１９−２５、ＯＲＴＨＯ　ＨＣＶ　Ｖｅｒｓｉｏｎ　３．０　ＥＬＩＳＡ　Ｔｅｓｔ　Ｓｙｓｔｅｍ，Ｏｒｔｈｏ−Ｃｌｉｎｉｃａｌ　Ｄｉａｇｎｏｓｔｉｃｓ，Ｒａｒｉｔａｎ，Ｎｅｗ　Ｊｅｒｓｅｙにおいて利用可能）もまた、マイクロタイタープレート上にコーティングした。処方物中の還元剤（ＤＴＴ）および界面活性剤（ＳＤＳ）の存在に起因して非立体構造性であると推測されるｃ２００抗原を、比較として用いた。
【０１５７】
免疫反応性を、２つの初期ＨＣＶセロコンバージョンパネルＰＨＶ９０４およびＰＨＶ９１４（Ｂｏｓｔｏｎ　Ｂｉｏｍｅｄｉｃａ，Ｉｎｃ．，Ｗｅｓｔ　Ｂｒｉｄｇｅｗａｔｅｒ，ＭＡから市販されているヒト血液サンプル）に対して試験した。これらの結果を表９に示す。これらのデータは、変性または直鎖形態のＮＳ３／４ａ（およびｃ２００）が、ＮＳ３／４ａコンホメーションエピトープと同じくらい早く初期セロコンバージョンパネルを検出しないことを示唆する。
【０１５８】
【表９】

コンホメーションエピトープの免疫反応性を、標準的な手順を用いて作製されたＮＳ３／４ａに対するモノクローナル抗体を用いても試験した。次いで、これらのモノクローナル抗体を、ＮＳ３／４ａおよび変性ＮＳ３／４ａならびにｃ２００抗原に対して、ＥＬＩＳＡフォーマットにおいて試験した。これらのデータは、抗ＮＳ３／４ａモノクローナル抗体が、表１０に示すセロコンバージョンパネルと同様の様式で、ＮＳ３／４ａおよび変性ＮＳ３／４ａと反応することを示す。この結果はまた、初期ｃ３３ｃセロコンバージョンパネルに対する反応性において類似するモノクローナル抗体が作製され得る場合、ＮＳ３／４ａが本質的に立体構造性であることのさらなる証拠を提供する。
【０１５９】
【表１０】

従って、新規のＨＣＶ検出アッセイを開示した。前述のことから、本発明の特定の実施形態が、例示の目的で本明細書中に記載されているが、本発明の意図および範囲から逸脱することなく種々の改変がなされ得ることが理解される。
【図面の簡単な説明】
【図１】
図１は、ＨＣＶゲノムの模式的表現であり、本発明のアッセイ試薬（タンパク質および抗体）が由来するポリタンパク質の種々の領域を示す。
【図２】
図２は、本発明の代表的な抗体／抗原組合せアッセイの模式図である。
【図３】
図３は、本発明のアッセイにおける使用のための代表的なＮＳ３／４ａコンホメーション抗原のアミノ酸配列を示す。１８２位の太文字のアラニンは、この位置に通常存在するネイティブなセリンに置換される。
【図４】
図４Ａ〜４Ｄは、本発明のアッセイにおける使用のための別の代表的なＮＳ３／４ａコンホメーション抗原のＤＮＡおよび対応するアミノ酸配列を示す。図４Ａ〜４Ｄの４０３位および４０４位のアミノ酸は、ＨＣＶ−１のネイティブなアミノ酸配列のＰｒｏのＴｈｒへの置換、およびＩｌｅのＳｅｒへの置換を表す。
【図５】
図５は、ｐｄ．ＨＣＶ１ａ．ｎｓ３ｎｓ４ａＰＩの構築の模式図である。
【図６】
図６は、ＭＥＦＡ　１２の模式的表現である。
【図７】
図７Ａ〜７Ｆは、ＭＥＦＡ　１２のＤＮＡおよび対応するアミノ酸配列を示す。
【図８】
図８は、ＭＥＦＡ　１２を使用する、本発明の代表的なイムノアッセイの模式図である。[0001]
(Technical field)
The present invention relates generally to the diagnosis of viruses. In particular, the invention relates to a combined antigen / antibody assay for accurately diagnosing hepatitis C virus infection.
[0002]
(Background of the Invention)
Hepatitis C virus (HCV) is the leading cause of parenteral non-hepatitis A, non-B hepatitis (NANBH), which is transmitted primarily through blood transfusion and sexual contact. This virus is present in 0.4-2.0% of blood donors. Chronic hepatitis develops in about 50% of infected individuals, and approximately 20% of these infected individuals develop cirrhosis, which often causes hepatocellular carcinoma. Therefore, the study and control of this disease is medically important.
[0003]
HCV was first identified and characterized as the cause of NANBH by Houghten et al. This sequence is known, as are the methods for obtaining the HCV viral genomic sequence. See, for example, International Publication Nos. WO 89/04669; WO 90/11089; and WO 90/14436. HCV has a 9.5 kb positive-sense, single-stranded RNA genome and is a member of the Flaviridae family of viruses. Based on phylogenetic analysis, at least six different related HCV genes have been identified (Simmonds et al., J. Gen. Virol. (1993) 74: 2391-2399). These viruses encode a single polyprotein with more than 3000 amino acid residues (Choo et al., Science (1989) 244: 359-369; Choo et al., Proc. Natl. Acad. Sci. USA (1991). 88: 2451-2455; Han et al., Proc. Natl. Acad. Sci. USA (1991) 88: 1711-1715). Polyproteins are processed into both structural and non-structural (NS) proteins simultaneously with and after translation.
[0004]
In particular, as shown in FIG. 1, some proteins are encoded by the HCV genome. The order and scientific names of the cleavage products of the HCV polyprotein are as follows: NH ₂ -C-E1-E2-p7-NS2-NS3-NS4a-NS4b-NS5a-NS5b-COOH. The first cleavage of the polyprotein involves three structural proteins (the N-terminal nucleocapsid protein (referred to as "core") and two envelope glycoproteins ("E1" (also known as E) and "E2" (also known as E2 / NS1). Known))) as well as catalyzed by host proteases, releasing non-structural (NS) proteins, including viral enzymes. The NS regions are called NS2, NS3, NS4, and NS5. NS2 is an integral membrane protein with proteolytic activity. NS2, either alone or in combination with NS3, cleaves the NS2-NS3 sissle bond, which in turn gives rise to the N-terminus of NS3 and increases the activity of both serine proteases and RNA helicases. Release large polyprotein containing NS3 protease serves to process the remaining polyprotein. Termination of polyprotein maturation is initiated by autocatalytic cleavage at the junction of NS3-NS4a, catalyzed by the NS3 serine protease. Subsequent NS3-mediated cleavage of the HCV polyprotein appears to be related to recognition of the polyprotein cleavage junction by the NS3 molecule of another polypeptide. In these reactions, NS3 releases the NS3 cofactor (NS4a), two proteins (NS4b and NS5a), and RNA-dependent RNA polymerase (NS5b).
[0005]
A number of general and specific polypeptides from HCV polyprotein useful as immunological and diagnostic reagents for HCV have been described. For example, Houghton et al., European Publication Nos. 318,216 and 388,232; Choo et al., Science (1989) 244: 359-362; Kuo et al., Science (1989) 244: 362-364; Houghton et al., Hepatology (1991). 14: 381-388; Chien et al., Proc. Natl. Acad. Sci. ScL USA (1992) 89: 10011-10015; Chien et al. Gastroent. Hepatol. (1993) 8: S33-39; Chien et al., International Publication No. WO 93/00365; Chien, D .; Y. , International Publication No. WO 94/01778. These publications generally provide an extensive background on the manufacture and use of HCV and immunological reagents for HCV polypeptides.
[0006]
Sensitive and specific methods for screening and identifying HCV carriers and blood or blood products contaminated with HCV provide important advances in pharmaceuticals. Post-transfusion hepatitis (PTH) developed in approximately 10% of transfused patients, and HCV was responsible for up to 90% of these cases. Reliable diagnostic and prognostic tools are needed for patient care and for the prevention and transmission of HCV by blood and blood products or by close personal contact. Accordingly, several assays have been developed for serodiagnosis of HCV infection. See, e.g., Choo et al., Science (1989) 244: 359-362; Kuo et al., Science (1989) 244: 362-364; Choo et al., Br. Med. Bull. (1990) 46: 423-441; Ebeling et al., Lancet (1990) 335: 982-983; van der Poel et al., Lancet (1990) 335: 558-560; van der Poel et al., Lancet (1991) 337: 317-. 319; Chien, D .; Y. See, International Publication No. WO 94/01778; Valenzuela et al., International Publication No. WO 97/44469; and Kashiwakuma et al., US Pat. No. 5,871,904.
[0007]
A significant problem encountered with some serum-based assays is that there is a significant interval (often more than 80 days) between infection and detection of the virus. The interval of this assay can create significant risks to transfusion recipients. To overcome this problem, nucleic acid-based tests (NAT) that detect viral RNA directly, and HCV core antigen tests that assay viral antigens instead of antibody responses, have been developed. See, for example, Kashiwakuma et al., U.S. Patent No. 5,871,904; Beld et al., Transfusion (2000) 40: 575-579.
[0008]
However, there remains a need for sensitive and accurate diagnostic and prognostic tools to provide adequate patient care and prevent HCV infection by blood and blood products or by close personal contact. I have.
[0009]
(Summary of the Invention)
The present invention is based, in part, on the finding that HCV seroconversion antibodies are typically anti-core and anti-NS-3 (helicase). Thus, the present invention provides a combined HCV core antigen and NS3 antibody assay that can detect both HCV antigen and HCV antibody present in a sample using a single solid matrix.
[0010]
Accordingly, in one embodiment, the invention relates to an immunoassay solid support comprising at least one HCV anti-core antibody and at least one isolated HCV NS3 / 4a epitope binding thereto. The antibody and NS3 / 4a epitope can be any of the molecules described herein. In addition, the solid support can include any of the multi-epitope fusion antigens described herein, such as a multi-epitope fusion antigen comprising the amino acid sequence shown in FIGS.
[0011]
In certain embodiments, the solid support comprises at least two HCV anti-core antibodies attached thereto. Further, the anti-core antibody can be a monoclonal antibody. Further, the NS3 / 4a epitope can be a conformational epitope (eg, a conformational NS3 / 4a epitope comprising the amino acid sequences shown in FIGS. 4A-4D).
[0012]
In another embodiment, the present invention relates to an immunoassay solid comprising at least two HCV anti-core monoclonal antibodies and at least one HCV NS3 / 4a conformational epitope (including the amino acid sequence shown in FIGS. 4-4D) binding thereto. Regarding the support.
[0013]
In yet a further embodiment, the invention is directed to a method for detecting HCV infection in a biological sample. The method comprises the following steps: (a) providing an immunoassay solid support as described above; (b) HCV antigen and HCV antibody (if present in the biological sample) are each at least Combining the biological sample with the solid support under conditions that allow it to bind to one anti-core antibody and the NS3 / 4a epitope; (c) conjugate to the solid support from step (b) Under conditions that the body forms, (i) a detectably labeled first antibody, wherein the detectably labeled first antibody is a detectably labeled HCV anti-core antibody, wherein the The labeled anti-core antibody is directed against an HCV core epitope that is different from the at least one anti-core antibody bound to the solid support); (ii) a product reactive with the NS3 / 4a epitope An antigen reactive with an HCV antibody from the biological sample; and (iii) a detectably labeled second antibody, wherein the detectably labeled second antibody is reactive with the antigen of (ii). And (d) detecting the complex formed between the antibody and the antigen (if present) as an indicator of HCV infection in the biological sample. The NS3 / 4a epitope can be a conformational epitope (eg, a conformational epitope having the NS3 / 4a sequence shown in FIGS. 4A-4D).
[0014]
In yet another embodiment, the invention is directed to a method for detecting HCV infection in a biological sample. The method comprises the steps of: (a) providing an immunoassay solid support as described above (having at least two HCV anti-core antibodies bound thereto); (b) HCV antigens and HCV antibodies (biological Combining the biological sample with the solid support under conditions that allow binding to the at least two anti-core antibodies and the NS3 / 4a epitope, respectively, if present in the biological sample); B.) On the solid support from step (b), under the conditions under which the complex is formed, (i) a detectably labeled first antibody, wherein the detectably labeled first antibody is A labeled HCV anti-core antibody, wherein the labeled anti-core antibody is directed against a different HCV core epitope than the anti-core antibody bound to the solid support); ii) an epitope from the c33c region of the HCV polyprotein fused to the hSOD amino acid sequence; and (iii) a detectably labeled second antibody, wherein the detectably labeled second antibody comprises hSOD. (D) reacting with the amino acid sequence); and (d) detecting the complex formed between the antibody and the antigen (if present) as an indicator of HCV infection in the biological sample Process. The NS3 / 4a epitope can be a conformational epitope (eg, a conformational epitope having the NS3 / 4a sequence shown in FIGS. 4A-4D).
[0015]
In any of the above embodiments, the anti-core antibody is directed against the N-terminal region of the HCV core antigen (eg, against amino acids 10-53 of HCV, numbered relative to the HCV1 polyprotein sequence). The resulting and / or detectably labeled HCV anti-core antibody is directed against the C-terminal region of the HCV core antigen (eg, amino acids 120-130 of HCV numbered relative to the HCV1 polyprotein sequence). obtain. In addition, antigens that react with HCV antibodies from a biological sample can be from the NS3 region (eg, an epitope from the c33c region of the HCV polyprotein) and can be fused to a human superoxide dismutase (hSOD) amino acid sequence. In this embodiment, the detectably labeled second antibody is reactive with the hSOD amino acid sequence.
[0016]
In another embodiment, the invention is directed to a method for detecting HCV infection in a biological sample. The method comprises the steps of: (a) providing an immunoassay solid support comprising two HCV anti-core monoclonal antibodies and a conformational epitope comprising the amino acid sequence shown in FIGS. 4A-4D; (b) Under conditions that allow the HCV antigen and HCV antibody (if present in the biological sample) to bind to at least two anti-core antibodies and the NS3 / 4a conformational epitope, respectively, Combining the solid support from step (b) with the solid support; and (i) a first antibody detectably labeled under conditions in which the complex is formed, wherein the first antibody is detectably labeled. The first antibody identified is a detectably labeled HCV anti-core antibody, wherein the labeled anti-core antibody binds to a solid support. At least two anti-core antibodies are directed against a different HCV core epitope); (ii) an epitope from the c33c region of the HCV polyprotein fused to the hSOD amino acid sequence; and (iii) detectably labeled. Adding a second antibody, wherein the detectably labeled second antibody is reactive with the hSOD amino acid sequence; and (d) between the antibody and the antigen (if present). Detecting the complex formed in the biological sample as an indicator of HCV infection.
[0017]
In certain embodiments, at least two anti-core antibodies are directed against the N-terminal region of the HCV core antigen (eg, against amino acids 10-53 of HCV, numbered for the HCV1 polyprotein). And detectably labeled HCV anti-core antibody is directed against the C-terminal region of the HCV core antigen (eg, against amino acids 120-130 of HCV numbered relative to the HCV1 polyprotein sequence). Is done.
[0018]
In another embodiment, the invention is directed to a method for detecting HCV infection in a biological sample. The method comprises the steps of: (a) providing an immunoassay solid support comprising a multi-epitope fusion antigen; (b) at least HCV antigen and HCV antibody (if present in a biological sample). Combining the biological sample with the solid support under conditions that allow binding to one anti-core antibody, the NS3 / 4a epitope and the multi-epitope fusion antigen; (c) the solid from step (b) The support is provided with a (i) detectably labeled first antibody under conditions in which the complex is formed, wherein the first detectably labeled antibody is a detectably labeled HCV anti-core antibody. And wherein the labeled anti-core antibody is directed against a different HCV core epitope than at least one anti-core antibody bound to a solid support); (ii) biology A first antigen and a second antigen that are reactive with HCV antibodies from the sample (reactive with the NS3 / 4a epitope and the multi-epitope fusion antigen, respectively); and (iii) a detectably labeled second antibody ( Wherein the detectably labeled second antibody is (ii) reactive with the antigen); and (d) is formed between the antibody and the antigen (if present). Detecting the complex as an indicator of HCV infection in a biological sample.
[0019]
As described above, the anti-core antibody can be directed against the N-terminal region of the HCV core antigen, and the first detectably labeled HCV anti-core antibody is directed against the C-terminal region of the HCV core antigen. Can be oriented. Further, the first antigen that reacts with HCV antibodies from a biological sample can include an epitope from the c33c region of an HCV polyprotein and can be fused to an hSOD amino acid sequence. In this situation, the detectably labeled second antibody is reactive with the hSOD amino acid sequence. In addition, a second antigen that reacts with HCV antibodies from a biological sample is an epitope from the c22 region of the HCV polyprotein (eg, Arg47 deletion and position 44, numbered relative to the HCV1 polyprotein sequence). Amino acids Lys of HCV polyprotein with Leu substitution for Trp in Escherichia coli ₁₀ ~ Ser ₉₉ Including epitopes). This epitope can be fused to the hSOD amino acid sequence. In that case, the detectably labeled second antibody is reactive with the hSOD amino acid sequence. Multi-epitope fusion antigens can include the amino acid sequences set forth in FIGS.
[0020]
In yet a further embodiment, the invention relates to a method of detecting HCV infection in a biological sample, comprising: (a) providing an immunoassay solid support, wherein the solid support comprises: Two HCV anti-core monoclonal antibodies, an HCV NS3 / 4a conformational epitope (containing the amino acid sequence set forth in FIGS. 4A-4D), and a multi-epitope fusion antigen (FIGS. 7A-7F) bound to this solid support. (B) at least two anti-core antibodies, the NS3 / 4a conformational epitope, and the multiplex when the HCV antigen and antibody are present in a biological sample. Combining the biological sample with a solid support under conditions that bind to each of the epitope fusion antigens; ( c) under complexing conditions, on the solid support from step (b), (i) a first detectably labeled antibody, wherein the first detectably labeled antibody is detected A possible labeled HCV anti-core antibody, wherein the labeled anti-core antibody is directed against a different HCV core epitope than at least two anti-core antibodies attached to a solid support) (Ii) an epitope from the c33c region of the HCV polyprotein fused to the hSOD amino acid sequence and an epitope from the c22 region of the HCV polyprotein fused to the hSOD amino acid sequence; and (iii) a second detectably labeled (D) (where present, the second detectably labeled antibody is reactive with the hSOD amino acid sequence). Detecting the complex between) antibody and antigen as an indicator of HCV infection in an object biological samples including,.
[0021]
In this embodiment, at least two anti-core antibodies are directed against the N-terminal region of the HCV core antigen (eg, against amino acids 10-53 of HCV (numbered for HCV1 polyprotein)). The resulting and detectably labeled HCV anti-core antibody is raised against the C-terminal region of the HCV core antigen (eg, against amino acids 120-130 of HCV (numbered for HCV1 polyprotein)). Be oriented. In addition, the epitope from the c22 region is the amino acid Lys of the HCV polyprotein, numbered for the HCV1 polyprotein. ₁₀ ~ Ser ₉₉ Where Arg47 is deleted and at position 44 Lue is replaced by Trp.
[0022]
In another embodiment, the invention relates to an immunodiagnostic test kit, comprising the immunoassay solid support described above and instructions for performing the immunodiagnostic test.
[0023]
In a still further embodiment, the invention relates to a method of producing an immunoassay solid support, the method comprising: (a) providing a solid support; and (b) at least one HCV anti-core antibody (eg, 1 One or more) and at least one isolated HCV NS3 / 4a epitope, and optionally, a multi-epitope fusion antigen, to a solid support. The anti-core antibody, NS3 / 4a epitope, and multiple epitope fusion antigen are as described above.
[0024]
In a further embodiment, the invention relates to multi-epitope fusion antigens, wherein the antigens have at least 80% sequence identity (e.g., 90% or more) to the amino acid sequence set forth in FIGS. Sequence identity), which specifically reacts with anti-HCV antibodies present in biological samples from HCV-infected individuals. In certain embodiments, the multi-epitope fusion antigen consists of the amino acid sequences shown in FIGS.
[0025]
In a further embodiment, the present invention provides a polynucleotide comprising a coding sequence for a multiple epitope fusion antigen as described above, a recombinant vector comprising the polynucleotide, a host cell transformed with the recombinant vector, and a recombinant multiple epitope. For a method of making a fusion antigen, the method comprises the steps of: (a) providing a population of host cells as described above; and (b) a multiple epitope fusion antigen encoded by a coding sequence present in the recombinant vector. Culturing this population of cells under conditions in which is expressed.
[0026]
These and other aspects of the invention will become apparent with reference to the following detailed description and accompanying drawings.
[0027]
(Detailed description of the invention)
The practice of the present invention will employ, unless otherwise indicated, conventional methods of chemistry, biochemistry, recombinant DNA technology and immunology, within the skill of the art. Such techniques are explained fully in the literature. For example, Fundamental Virology, 2nd Edition, Volumes I and II (BN Fields and DM Knipe, eds.); Handbook of Experimental Immunology, Volumes I-IV (DM Weir and C.C. T. Blackwell, Blackwell Scientific Publications); E. FIG. Creighton, Proteins: Structures and Molecular Properties (WH Freeman and Company, 1993); L. Lehninger, Biochemistry (Worth Publishers, Inc., current addition); Sambrook et al., Molecular Cloning: A Laboratories Manual (2nd ed., USA). , Inc.).
[0028]
As used in this specification and the appended claims, the singular forms "a,""an," and "the" include plural references unless the content clearly dictates otherwise. You have to be careful. Thus, for example, a reference to "antigen" includes a mixture of two or more antigens, and so forth.
[0029]
The following amino acid abbreviations are used throughout this specification:
Alanine: Ala (A) Arginine: Arg (R)
Asparagine: Asn (N) Aspartic acid: Asp (D)
Cysteine: Cys (C) Glutamine: Gln (Q)
Glutamic acid: Glu (E) Glycine: Gly (G)
Histidine: His (H) Isoleucine: Ile (I)
Leucine: Leu (L) Lysine: Lys (K)
Methionine: Met (M) Phenylalanine: Phe (F)
Proline: Pro (P) Serine: Ser (S)
Threonine: Thr (T) Tryptophan: Trp (W)
Tyrosine: Tyr (Y) Valine: Val (V)
(I. Definition)
In describing the present invention, the following terms will be used, and are intended to be defined as indicated below.
[0030]
The terms "polypeptide" and "protein" refer to a polymer of amino acid residues. And it is not limited to the minimum length of the product. Thus, peptides, oligopeptides, dimers, multimers and the like are included in this definition. Both full length proteins and fragments thereof are included in this definition. The term also includes post-expression modifications of the polypeptide (eg, glucosylation, acetylation, phosphorylation, etc.). Further, for the purposes of the present invention, a "polypeptide" is a modification (e.g., deletion, addition and substitution (generally conservative in character) of the native sequence to the native sequence as long as the desired activity of the protein is maintained. A)). These modifications may be deliberate, such as through site-directed mutagenesis, or may be accidental, such as through errors due to mutations or PCR amplification of the host producing the protein. obtain.
[0031]
An HCV polypeptide is a polypeptide derived from an HCV polyprotein, as described above. The polypeptide need not be physically derived from HCV but can be produced synthetically or recombinantly. Further, the polypeptide may be from any of a variety of HCV strains and isolates, including, but not limited to, any of the isolates from strains 1, 2, 3, or 4 of HCV. Many conserved and variable regions are known in these strains, and in general, the amino acid sequences of the epitopes derived from these regions, when the two sequences are aligned, have a high degree of sequence homology (eg, , Greater than 30%, preferably greater than 40%). Thus, for example, the term "NS3 / 4a" polypeptide refers to native NS3 / 4a from any of a variety of HCV strains, as well as NS3 / 4a analogs, muteins and immunogenic fragments as further defined below. Say. The complete genotype of many of these strains is known. See, for example, U.S. Patent No. 6,150,087 and GenBank accession numbers AJ238800 and AJ238799.
[0032]
The terms "analog" and "mutein" are defined as biologically active derivatives of the reference molecule or fragments of such derivatives, which are those that have a desired activity, such as immunoreactivity, in the assays described herein. Maintain). In general, the term "analog" has one or more amino acid additions, substitutions (generally conservative in nature), and / or deletions with respect to the native molecule, unless the alteration destroys immunogenic activity. A compound having a native polypeptide sequence and structure. The term "mutein" refers to a peptide having one or more peptidomimetics ("peptoids") and is described, for example, in International Publication No. WO 91/04282. Preferably, the analog or mutein has at least the same immunological activity as the native molecule. Methods for making polypeptide analogs and muteins are known in the art and are described further below.
[0033]
Particularly preferred analogs include substitutions that are conservative in nature (ie, those substitutions that occur within the family of amino acids that involve their side chains). Specifically, amino acids are generally classified into four families: (1) acidic--aspartic acid and glutamic acid; (2) basic--lysine, arginine, histidine; (3) nonpolar--alanine. Valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) uncharged and polar--glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. Phenylalanine, tryptophan, and tyrosine are often classified as aromatic amino acids. For example, a single substitution of leucine for isoleucine or valine, of aspartic acid for glutamic acid, for a threonine for serine, or a similar conservative substitution of an amino acid for a structurally related amino acid, would result in a loss of biological activity. Has no major effect, which is reasonably predictable. For example, a polypeptide of interest can have up to about 5-10 conservative amino acid substitutions or non-conservative amino acid substitutions, or up to about 15-25 conservative amino acid substitutions or as long as the desired function of the molecule remains intact. It may have non-conservative amino acid substitutions, or any integer between 5 and 25 conservative or non-conservative amino acid substitutions. One of skill in the art can readily determine the regions of a molecule of interest that can tolerate variation by referencing Hopp / Woods and Kyte-Doolittle plots, well known in the art.
[0034]
"Fragment" intends a polypeptide consisting of only part of the intact full-length polypeptide sequence and structure. This fragment may contain a C-terminal deletion and / or an N-terminal deletion of the native polypeptide. An "immunogenic fragment" of a particular HCV protein generally comprises at least about 5-10 contiguous amino acid residues of the full length molecule, preferably at least about 15-25 contiguous amino acid residues of the full length molecule, and most preferably Contains at least about 20-50 or more contiguous amino acid residues of the molecule (which define the epitope), or contains any integer number of contiguous amino acid residues between 5 amino acids and the full length sequence, However, the fragment in question retains immunoreactivity in the assays described herein. For example, preferred immunogenic fragments include, for example, amino acids 10-45, 10-53, 67-88, and 120-130 of the polyprotein, epitope 5-1-1 (in the NS3 region of the viral genome) and HCV poly An HCV core comprising defined epitopes from the El, E2, c33c (NS3), c100 (NS4), NS3 / 4a and NS5 regions of the protein, and any of the various epitopes of the protein identified from the HCV polyprotein. Fragments, but are not limited to these. See, for example, Chien et al., Proc. Natl. Acad. Sci. USA (1992) 89: 10011-10015; Chien et al. Gastroent. Hepatol. (1993) 8: S33-39; Chien et al., International Publication No. WO 93/00365; Chien, D .; Y. See, International Publication No. WO 94/01778; U.S. Patent Nos. 6,150,087 and 6,121,020.
[0035]
As used herein, the term "epitope" refers to a sequence of at least about 3-5, preferably about 5-10 or 15, and no more than about 1000 amino acids (or any integer number of amino acids in between). This, by itself or as part of a larger sequence, defines a sequence that binds to an antibody produced in response to such a sequence. There is no critical upper limit on the length of the fragment, and the fragment may include a nearly full-length protein sequence, or even a fusion protein comprising two or more epitopes from an HCV polyprotein. Epitopes for use in the present invention are not limited to polypeptides having the exact sequence of a portion of the parent protein from which they are derived. Indeed, the viral genome contains several variable domains that are in constant flux and exhibit a relatively high degree of variability between isolates. Thus, the term "epitope" encompasses sequences identical to the native sequence, as well as modifications to the native sequence, such as deletions, additions and substitutions, which are generally conservative in nature.
[0036]
The region of a given polypeptide that contains an epitope can be identified using any number of epitope mapping techniques well known in the art. See, for example, Epitope Mapping Protocols, Methods in Molecular Biology, Vol. 66 (Edited by Glenn E. Morris, 1996) Humana Press, Totowa, New Jersey. For example, a linear epitope can be used, for example, to simultaneously synthesize a large number of peptides (these peptides corresponding to a portion of a protein molecule) on a solid support and still attach these peptides to the solid support. Can be determined by reacting these peptides with antibodies. Such techniques are known in the art and are described, for example, in: US Pat. No. 4,708,871; Geysen et al. (1984) Proc. Natl. Acad. Sci. ScL USA 81: 3998-4002; Geysen et al. (1985) Proc. Natl. Acad. Sci. USA 82: 178-182; Geysen et al. (1986) Molec. Immunol. 23: 709-715. Using such techniques, a number of epitopes for HCV have been identified. See, for example, Chien et al., Virtual Hepatitis and Liver Disease (1994) pp. 320-324, and further below. Similarly, conformational epitopes are readily identified by determining the spatial conformation of amino acids, for example, by X-ray crystallography and two-dimensional nuclear magnetic resonance. See, for example, Epitope Mapping Protocols (supra). The antigenic region of the protein can also be determined using standard antigenicity and hydrophobicity hydrophilicity index plots (eg, those calculated using the Omiga version 1.0 software program available from Oxford Molecular Group). Can be identified. This computer program is based on the Hopp / Woods method for determining antigenicity profiles (Hopp et al., Proc. Natl. Acad. Sci USA (1981) 78: 3824-3828), and Kyte for hydrophobicity index plots. -Use the Doolittle technology (Kyte et al., J. Mol. Biol. (1982) 157: 105-132).
[0037]
As used herein, the term "conformational epitope" refers to a full-length protein, or an analog or analog thereof, of a full-length native protein having structural features native to the amino acid sequence encoding the epitope. A part of mutein. Native structural features include, but are not limited to, glycosylation and three-dimensional structure. The length of the epitope defining sequence can be subject to wide variations. This is because these epitopes are thought to be formed by the three-dimensional shape (eg, folding) of the antigen. Thus, the amino acids that define an epitope can be relatively small in number, but can be widely distributed along the length of a molecule (or even on different molecules, such as in the case of a dimer), resulting in a more accurate epitope Be conformed. The portion of the antigen between the residues that define the epitope may not be important for the conformational structure of the epitope. For example, deletions or substitutions of these intervening sequences affect the conformational epitope as long as sequences important for epitope conformation (eg, cysteines involved in disulfide bonds, glycosylation sites, etc.) are maintained. You don't have to.
[0038]
Conformational epitopes present in the NS3 / 4a region are easily identified using the methods discussed above. In addition, the presence or absence of a conformational epitope in a given polypeptide can be determined by screening the antigen of interest using an antibody (polyclonal or monoclonal serum against the conformational epitope) and determining its reactivity with a linear epitope. It can be readily determined by comparing to the reactivity of the denatured version of the antigen, which retains only (if present) In such screens using polyclonal antibodies, it may be advantageous to first absorb the polyclonal sera with the denatured antigen and observe whether the antibody against the antigen of interest is retained. In addition, in the case of NS3 / 4a, the molecule that preserves the native conformation also has protease enzyme activity and, optionally, helicase enzyme activity. Such activity can be detected using an enzymatic assay, as described further below.
[0039]
Preferably, the conformational epitope is recombinantly produced and expressed, for example, in a cell that can be extracted under conditions that preserve its desired structural characteristics without denaturation of the epitope. Such cells include bacterial cells, yeast cells, insect cells, and mammalian cells. Expression and isolation of recombinant conformational epitopes from HCV polyprotein is described, for example, in International Publication Nos. WO 96/04301, WO 94/01778, WO 95/33053, WO 92/08734. Alternatively, it is possible to express the antigen and further regenerate the protein after recovery. It is also understood that chemical synthesis can also provide a conformational antigen mimitope that cross-reacts with the "native" antigen conformational epitope.
[0040]
As used herein, the term "multi-epitope fusion antigen" or "MEFA" intends a polypeptide in which multiple HCV antigens are part of a single, continuous chain of amino acids, where the chain is , Does not exist in nature. HCV antigens may be directly linked to each other by peptide bonds, or may be separated by intervening amino acid sequences. Fusion antigens may also include sequences foreign to the HCV polyprotein. Further, the HCV sequences present may be from multiple genotypes and / or may be HCV isolates. Examples of specific MEFAs for use in the immunoassays of the invention are described in detail, for example, in International Publication No. WO 97/44469, and are described further below.
[0041]
"Antibody" intends a molecule that specifically binds to a polypeptide of interest by chemical or physical means. Thus, HCV core antibodies are molecules that specifically bind to HCV core proteins. As used herein, the term “antibody” refers to both polyclonal and monoclonal preparations, as well as the following: hybrid (chimeric) antibody molecules (eg, Winter et al. (1991) Nature 349: 293-299; And U.S. Patent No. 4,816,567); F (ab ') ₂ And F (ab) fragments; Fv molecules (non-covalent heterodimers, see, eg, Inbar et al. (1972) Proc Natl Acad Sci USA 69: 2659-2662; and Ehrlich et al. (1980) Biochem 19: 4091-4096). Single-chain Fv molecules (sFv) (see, for example, Huston et al. (1988) Proc Natl Acad Sci USA 85: 5879-5883); dimeric and trimeric antibody fragment constructs; minibodies (See, e.g., Pack et al. (1992) Biochem 31: 1579-1584; Cumber et al. (1992) J Immunology 149B: 120-126); humanized antibody molecules (e.g., Ri). (see chmann et al. (1988) Nature 332: 323-327; Verhoeyan et al. (1988) Science 239: 1534-1536; and British Patent Publication No. GB 2,276,169 published September 21, 1994); As well as antibodies derived from any functional fragment derived from such a molecule, wherein such fragments retain the immunological binding properties of the parent antibody molecule.
[0042]
As used herein, the term "monoclonal antibody" refers to an antibody composition having a homogeneous antibody population. The term is not limited with respect to the species or source of the antibody, nor is it intended to be limited by the method by which it is made. Thus, the term encompasses antibodies obtained from mouse hybridomas, as well as human monoclonal antibodies obtained using humans rather than mouse hybridomas. See, for example, Cote et al., Monclonal Antibodies and Cancer Therapy, Alan R.S. Liss, 1985, p. See 77.
[0043]
A “recombinant” protein is a protein that has the desired activity and has been prepared by the recombinant DNA techniques described herein. Generally, the gene of interest is cloned and then expressed in a transformed organism, as described further below. The host organism expresses the foreign gene under expression conditions to produce a protein.
[0044]
"Isolated" when referring to a polypeptide is that the indicated molecule is separated from the entire organism in which it is found in nature and is separate or substantially identical to other macromolecules of the same type. It means that it exists in the absence of a physical. With respect to a polynucleotide, the term "isolated" refers to a nucleic acid molecule that lacks all or part of the sequence normally associated with the polynucleotide in nature; or has a heterologous sequence that exists in nature but is related to the sequence. Sequence; or a molecule dissociated from a chromosome.
[0045]
By "equivalent antigenic determinant" is meant an antigenic determinant from a different subspecies or strain of HCV (eg, HCV strain 1, 2 or 3). More specifically, epitopes (eg, 5-1-1) are known, and such epitopes vary between strains 1, 2, and 3. Thus, epitopes 5-1-1 from three different strains are equivalent antigenic determinants and are therefore "copies" even if their sequences are not identical. In general, the amino acid sequences of equivalent antigenic determinants exhibit a high degree of sequence homology (eg, greater than 30%, preferably greater than 40%, amino acid sequence homology) when the two sequences are aligned. Have.
[0046]
"Homology" refers to the percentage of similarity between two polynucleotide or two polypeptide moieties. The two DNA sequences, or two polypeptide sequences, are such that the sequences are at least about 50%, preferably at least about 75%, more preferably at least about 80% -85%, over a molecule of defined length, Preferably "substantially homologous" to each other if they exhibit at least about 90%, and most preferably at least about 95% to 98% sequence similarity. As used herein, substantially homologous also refers to sequences that show complete identity to a particular DNA or polypeptide sequence.
[0047]
In general, "identity" refers to the exact nucleotide-to-nucleotide or amino acid-to-amino acid correspondence of two polynucleotides or polypeptide sequences, respectively. The percent identity is determined by aligning the sequences, counting the exact number of matches between the two aligned sequences, dividing by the length of the shorter sequence, and multiplying the result by 100. It can be determined by direct comparison of sequence information between two molecules.
[0048]
Computer programs that are readily available (e.g., ALIGN, Dayhoff, MO, Ed. Atlas of Protein Sequence and Structure MO Edition, Dayhoff, Supplement 5, 3: 353-358, National biomedical Federation, National Biotechnology, Washington, D.C.). This adapts the local homology algorithm of Smith and Waterman Advances in Appl. Math. 2: 482-489, 1981) for peptide analysis)) to aid in similarity or identity analysis. Can be done. Programs for determining nucleotide sequence similarity and identity (eg, BESTFIT, FASTA and GAP programs, which also depend on the Smith and Waterman algorithms), are available from Wisconsin Sequence Analysis Package, Version 8 (Genetics Computer). , Madison, WI). These programs are readily used with the default parameters recommended by the manufacturer and described in the Wisconsin Sequence Analysis Package referenced above. For example, the percent homology of a particular nucleotide sequence to a reference sequence can be determined using the Smith and Waterman homology algorithm with a default scoring table and gap penalties for six nucleotide positions.
[0049]
Another method of establishing the percentage of similarity in the context of the present invention is copyrighted by the University of Edinburgh and is described in John F. Collins and Shane S.M. Developed by Sturrok and manufactured by IntelliGenetics, Inc. The use of the MPSRCH package of programs distributed by (Mountain View, CA). From this suite of packages, the Smith-Waterman algorithm may be used, where the default parameters are used for the scoring table (eg, 12 gap open penalties, 1 gap extension penalty, and 6 gap extension penalties). gap). From the data generated, the "match" value reflects "sequence similarity." Other suitable programs for calculating percent identity or similarity between sequences are generally known in the art; for example, another alignment program is BLAST used with default parameters. is there. For example, BLASTN and BLASTP can be used using the following default parameters: genetic code = standard; filter = none; strand = both; cutoff = 60; expected = 10; Matrix = BLOSUM62; (Descriptions) = 50 sequences; Sort = HIGH SCORE; Database (Database) = No degeneracy, GenBank + EMBL + DDBJ + PDB + GenBank CDS translations + Swiss protein + Suppositate + PIR. Details of these programs can be found at the following internet address: http: // www. ncbi. nlm. gov / cgi-bin / BLAST.
[0050]
Alternatively, homology refers to hybridization of polypeptides under conditions that form stable duplexes between homologous regions, followed by digestion with a single-strand-specific nuclease, and determination of the size of the digested fragment. Can be determined by DNA sequences that are substantially homologous can be identified in Southern hybridization experiments, for example, under stringent conditions, as defined for that particular system. Defining appropriate hybridization conditions is within the skill of the art. See, eg, Sambrook et al., Supra; DNA Cloning, supra; Nucleic Acid Hybridization, supra.
[0051]
A “coding sequence” or sequence that “encodes” a selected polypeptide, when placed under the control of appropriate regulatory sequences, is transcribed (in the case of DNA) and translated into a polypeptide in vitro or in vivo. (In the case of mRNA) a nucleic acid molecule. The boundaries of the coding sequence are determined by a start codon at the 5 '(amino) terminus and a translation stop codon at the 3' (carboxy) terminus. A transcription termination sequence may be located 3 'to the coding sequence.
[0052]
"Operably linked" refers to an arrangement of elements wherein the components so described are configured to perform their desired function. Thus, a given promoter operably linked to a coding sequence may result in the expression of the coding sequence in the presence of appropriate transcription factors and the like. A promoter need not be contiguous with a coding sequence so long as it functions to direct the expression of the coding sequence. Thus, for example, an intervening, untranslated but transcribed sequence may be present between the promoter sequence and the coding sequence (so that introns may be transcribed), and the promoter sequence may still be "operable" in the coding sequence. Linked to ".
[0053]
"Control element" refers to a polynucleotide sequence that aids in the expression of a coding sequence to which it is linked. The term includes promoters, transcription termination sequences, upstream regulatory domains, polyadenylation signals, untranslated regions (including 5'-UTR and 3'-UTR), and, where appropriate, leader sequences and enhancers (which are collectively Providing transcription and translation of the coding sequence in the host cell).
[0054]
As used herein, a "promoter" is a DNA regulatory region capable of binding RNA polymerase in a host cell and initiating transcription of a downstream (3 'direction) coding sequence operably linked thereto. It is. For purposes of the present invention, a promoter sequence contains the minimum number of bases or elements necessary to initiate transcription of a gene of interest at a detectable level above background. The promoter sequence has a transcription initiation site, as well as a protein binding domain (consensus sequence) responsible for binding RNA polymerase. Eukaryotic promoters will often, but not always, contain "TATA" boxes and "CAT" boxes.
[0055]
The control sequence directs the transcription of the coding sequence in a cell when the RNA polymerase binds to the promoter sequence and transcribes the coding sequence into mRNA, which is then translated into the polypeptide encoded by the coding sequence. Is done.
[0056]
"Expression cassette" or "expression construct" refers to an assembly capable of directing the expression of a sequence or gene of interest. The expression cassette contains control elements as described above (eg, a promoter operably linked to the sequence or gene of interest (to direct their transcription)), and often also contains a polyadenylation sequence. including. In certain embodiments of the invention, the expression cassettes described herein can be included in a plasmid construct. In addition to the components of the expression cassette, the plasmid construct may also include one or more selectable markers, a signal that allows the plasmid construct to be present as single-stranded DNA (eg, an origin of replication of M13), at least one. Multicloning sites, and "mammalian" origins of replication (eg, the SV40 origin of replication or the adenovirus origin of replication).
[0057]
As used herein, "transformation" refers to a host of exogenous polynucleotide, regardless of the method used for insertion (eg, transformation by direct uptake, transfection, infection, etc.). Refers to insertion into cells. See further below for specific methods of transfection. The exogenous polynucleotide can be maintained as a non-integrated vector (eg, episome) or can be integrated into the host genome.
[0058]
"Host cell" refers to a cell that has been, or can be, transformed by an exogenous DNA sequence.
[0059]
"Common solid support" refers to a single solid substrate to which HCV polypeptides used in a subject immunoassay are bound by non-covalent means, such as covalent or hydrophobic adsorption.
[0060]
By "immunological response" is meant that the antigen of interest specifically reacts with anti-HCV antibodies present in a biological sample from an individual infected with HCV.
[0061]
"Immune complex" intends the combination formed when an antibody binds to an epitope on an antigen.
[0062]
As used herein, a "biological sample" refers to a sample of tissue or fluid isolated from a subject, including, but not limited to, blood, Plasma, serum, feces, urine, bone marrow, bile, cerebrospinal fluid, lymph, skin samples, the following external secretions (skin, respiratory tract, intestinal tract, and genitourinary tract), tears, saliva, milk, blood cells, organs, raw Samples of in vitro cell culture constructs, including, but not limited to, test materials, and also conditioned media obtained from the growth of cells and tissues in culture media (eg, recombinant cells, and cellular components).
[0063]
As used herein, the terms "label" and "detectable label" refer to a detectable molecule, including but not limited to: radioactive isotopes, fluorescent agents, chemiluminescent Chemiluminescers, luminophores, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, luminophores, dyes, metal ions, metal sols, ligands (eg, biotin, streptavidin or hapten), and the like. The term "fluorescent agent" refers to a substance or a part thereof that can show fluorescence in a detectable range. Particular examples of labels that can be used in the present invention include, but are not limited to, horseradish peroxidase (HRP), fluorescein, FITC, rhodamine, dansyl, umbelliferone, dimethylacridinium ester ( DMAE), Texas Red, Luminol, NADPH and α-β-galactosylase.
[0064]
(II. Embodiment of the Invention)
Before describing the present invention in detail, it is to be understood that this invention is not limited to particular formulas, which itself can, of course, vary. It should also be understood that the techniques used herein are for the purpose of describing particular embodiments of the present invention only and are not intended to be limiting.
[0065]
Although numerous compositions and methods similar or equivalent to those described herein can be used in the practice of the present invention, preferred materials and methods are described herein. Has been described.
[0066]
As mentioned above, the present invention is based on the discovery of new diagnostic methods for accurately detecting early HCV infection. This method relies on the identification and use of highly immunogenic HCV antibodies and HCV antigens, which are present during the early stages of seroconversion of HCV, thereby increasing detection accuracy. , And reduce the occurrence of false results. This method can be conveniently performed in a single assay format.
[0067]
More specifically, the assay comprises one or more HCV anti-core antibodies (directed to either the same or different HCV core epitopes) and the NS3 / 4a region of the HCV polyprotein. Performed on a solid support to which the epitope is attached. Examples of specific anti-core antibodies useful in the present invention include, but are not limited to: between amino acids 10-53; between amino acids 10-45; amino acids 67-88. An antibody molecule, such as a monoclonal antibody, directed against an epitope in the core region found between amino acids 120-130, or, for example, against any of the core epitopes identified below. Antibodies: Houghton et al., US Patent No. 5,350,671; Chien et al., Proc. Natl. Acad. Sci. USA (1992) 89: 10011-10015; Chien et al. Gastroent. Hepatol. (1993) 8: S33-39; Chien et al., International Publication No. WO 93/00365; Chien, D .; Y. And International Patent Publication Nos. WO 94/01778; and commonly owned U.S. Patent Application Serial Nos. 08 / 403,590 and 08 / 444,818.
[0068]
The NS3 / 4a region of the HCV polyprotein has been described, and its amino acid sequence and overall structure of this protein are described, for example, in Yao et al., Structure (November 1999) 7: 1353-1363; Sali et al., Biochem. (1998) 37: 3392-3401; and Bartensschlager, R .; , J. et al. Viral Hepat. (1999) 6: 165-181. See also Dasmahapatra et al., U.S. Patent No. 5,843,752. The immunoassays of the present invention utilize at least one conformational epitope from the NS3 / 4a region, which is present in a conformation as found in naturally occurring HCV particles or their infection products, to provide a protease, Helicase enzyme activity normally exhibited by the NS3 / 4a gene product and / or retention of antigen-antibody immunoreactivity in biological samples from HCV-infected subjects, and epitope immunoreactivity in antigen degeneration Becomes evident. For example, the conformational epitope can be modified by heating, extremely acidic or basic pH changes, or by adding known organic modifications (eg, dithiothreitol (DTT) or a suitable detergent). Can be collapsed. See, e.g., Protein Purification Methods, a practical approach (E.L.V. Harris and S. Angal, eds., IRL Press), see Denatured Products Compared to Products Not Treated as Above.
[0069]
Protease and helicase activities can be determined using standard enzyme assays well known in the art. For example, protease activity can be determined by using assays well known in the art. See, for example, Takeshita et al., Anal. Biochem. (1997) 247: 242-246; Kakiuchi et al. Biochem. (1997) 122: 749-755; Sali et al., Biochemistry (1998) 37: 3392-3401; Cho et al. Virol. Meth. (1998) 72: 109-115; Cerretani et al., Anal. Biochem. (1999) 266: 192-197; Zhang et al., Anal. Biochem (1999) 270: 268-275; Kakiuchi et al. Viol. Meth. (1999) 80: 77-84; Fowler et al. Biomol. Screen. (2000) 5: 152-158; and Kim et al., Anal. Biochem. (2000) 284: 42-48. A particularly convenient assay for testing protease activity is described in the Examples below.
[0070]
Similarly, helicase activity assays are well known in the art, and helicase activity of the NS3 / 4a epitope can be determined, for example, using the following: See, for example, Hsu et al., Biochem. Biophys. Res. Commun. (1998) 253: 594-599; an ELISA assay; Kyono et al., Anal. Biochem. (1998) 257: 120-126, a scintillation assay system; see, for example, Hicham et al., Antiviral Res. (2000) 46: 181-193 and Kwang et al., Methods Mol. Med. (2000) 24: 97-116; high throughput screening assays; as well as other assay methods known in the art. See, for example, Khu et al. Viol. (2001) 75: 205-214; Utama et al., Virology (2000) 273: 316-324; Paolini et al. Gen. Viol. (2000) 81: 1335-1345; Preugshat et al., Biochemistry (2000) 39: 5174-5183; Preugshat et al., Methods Mol. Med. (1998) 19: 353-364; and Hesson et al., Biochemistry (2000) 39: 2619-2625.
[0071]
The length of the antigen is sufficient to maintain an immunoreactive conformational epitope. Often, the polypeptide containing the antigen used is almost full-length, but the polypeptide can also be cleaved, for example, to increase solubility or improve secretion. Generally, the conformational epitopes found in NS3 / 4a are expressed as recombinant polypeptides in cells, and the polypeptides provide the desired form of the epitope as described in detail below.
[0072]
Representative amino acid sequences for NS3 / 4a polypeptides are shown in FIG. 3 and FIGS. The bold alanine present at position 182 in FIG. 3 is replaced with the native serine found at this position to prevent autocatalysis of the otherwise possible molecule. The amino acid sequence shown at positions 2 to 686 in FIGS. 4A to 4D corresponds to amino acids 1027 to 1711 of HCV-1. The start codon (ATG) encoding Met is shown as position 1. Furthermore, Thr which is normally present at position 1428 of HCV-1 (amino acid position 403 in FIG. 4) is mutated to Pro, and Ser which is normally present at position 1429 of HCV-1 (amino acid position 404 in FIG. 4) is , Ile. However, as long as this epitope is produced using a method that preserves or restores its native conformation such that protease activity and, optionally, helicase activity is retained, it will have a Met at the N-terminus, Either the native sequence, with or without the Met at the N-terminus, or a depicted analog, or other analogs and fragments, can be used in the assays of the invention. No. 5,843,752 and Zhang et al., US Pat. No. 5,990,276, both of which describe analogs of NS3 / 4a.
[0073]
The NS3 / 4a NS3 protease is found at about positions 1027 to about 1207 (positions 2 to 182 in FIG. 4) numbered for HCV-1. The structure and active site of the NS3 protease are known. See, for example, De Francesco et al., Antivir. Ther. (1998) 3: 99-109; Koch et al., Biochemistry (2001) 40: 631-640. Changes to the native sequence that are usually resistant are changes other than at the active site of the molecule. In particular, it is desirable that amino acid positions 1 to 155 or positions 2 to 155 in FIG. 4 are retained and are hardly substituted or only conservative substitutions are made. Amino acids present above 155 are more resistant to larger changes. Furthermore, when fragments of the NS3 / 4a sequence found in FIG. 4 are used, these fragments generally have at least amino acids 1 to 155 or 2 to 3 with or without a Met at the N-terminus. Position 155, preferably comprises amino acids 1 to 175 or 2 to 175, and most preferably amino acids 1 to 182 or 2 to 182. This helicase domain is found at about positions 1193 to about 1657 of HCV-1 (positions 207 to 632 in FIG. 4). Thus, if helicase activity is desired, the position of this molecule will be maintained with little or only conservative changes. One skilled in the art can readily determine other areas that will tolerate changes based on the known structure of NS3 / 4a.
[0074]
The solid support may also contain other antigens. For example, multiple epitope fusion antibodies (referred to as "MEFAs"), such as those described in International Publication No. WO 97/44469, can be bound to a solid support for use in the assays of the present invention. Such MEFAs contain multiple epitopes derived from two or more of the various viral regions shown in FIG. 1 and Table 1. In particular, as shown in FIG. 1 and Table 1, the cleavage of the HCV polyprotein ₂ -Produce at least 10 different products in the order core-E1-E2-p7-NS2-NS3-NS4a-NS4b-NS5a-NS5b-COOH. This core polypeptide is present at positions 1 to 191 numbered relative to HCV-1 (for the HCV-1 genome, Choo et al. (1991) Proc. Natl. Acad. Sci. USA 88: 2451-). 2455). The polypeptide is further processed to produce an HCV polypeptide comprising about amino acids 1 to about 173. The envelope polypeptides (E1 and E2) are present at about 192 to about 383 and about 384 to about 746, respectively. The P7 domain is found at about position 747 to about position 809. NS2 is a complete membrane protein with proteolytic activity and is found at about position 810 to about 1026 of this polyprotein. NS2 cleaves the NS2-NS3 single bond, either alone or in combination with NS3 (found at about position 1027 to about position 1657), and then generates the NS3 N-terminus And releases large polyproteins containing both serine protease activity and RNA helicase activity. This NS3 protease (found at about position 1027 to about position 1207) serves to process the remaining polyprotein. Helicase activity is found at about position 1193 to about position 1657. Completion of polyprotein maturation is initiated by autocatalytic cleavage of the NS3-NS4a bond catalyzed by the NS3 serine protease. Subsequent NS-3 mediated cleavage of the HCV polyprotein appears to be involved in the recognition of the cleavage of the polyprotein by the NS3 molecule of another polypeptide. In these reactions, NS3 is an NS3 cofactor (NS4a found at about position 1658 to about 1711), two proteins (NS4b found at about 1712 to about 1972, and about 1973 to about 2420). NS5a), as well as RNA-dependent RNA polymerase (NS5b found at about positions 2421 to about 3011).
[0075]
[Table 1]

^* Numbered for HCV-1. Choo et al. (1991) Proc. Natl. Acad. Sci. ScL USA 88: 2451-2455.
[0076]
Multiple HCV antigens are part of a single, continuous chain of amino acids whose chains are not naturally occurring. Thus, the linear order of the epitopes is different from their linear order in the genome where the epitopes occur. The linear order of the MEFA sequence for use herein is preferably arranged for optimal antigenicity. Preferably, these epitopes are from more than one HCV strain, thus providing the added ability to detect multiple HCV strains in one assay. Thus, MEFAs for use herein may include various immunogenic regions from the polyproteins described above. In addition, proteins resulting from a frameshift in the core region of the polyprotein (eg, proteins as described in International Publication No. WO 99/63941) can be used in MEFA. If desired, at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more of one or more epitopes from the HCV polyprotein can occur in the fusion protein.
[0077]
For example, an epitope from the hypervariable region of E2 (eg, the region spanning amino acids 384-410 or 390-410) can be included in a MEFA antigen. Particularly effective E2 epitopes include consensus sequences from this region (e.g., the consensus sequence Gly-Ser-Ala-Ala-Arg-Thr-Thr-Ser-Gly-Phe-Val-Ser-Leu-Phe-Ala-Pro- Epitope containing Gly-Ala-Lys-Gln-Asn, which represents a consensus sequence of amino acids 390-410 of the HCV type 1 genome. Representative E2 epitopes present on MEFAs of the present invention may include hybrid epitopes spanning amino acids 390-444. Such a hybrid E2 epitope may include a consensus sequence representing amino acids 390-410 fused to the native amino acid sequence of amino acids 411-444 of HCV E2.
[0078]
Further, the antigen may be from various HCV strains. Multiple viral strains of HCV are known, and epitopes from any of these strains can be used in the fusion protein. It is well known that any given species of an organism will vary from individual organism to individual organism, and that certain organisms such as viruses will have many different strains. For example, as explained above, HCV contains at least six genotypes. Each of these genotypes contains equivalent antigenic determinants. More specifically, each strain contains a number of antigenic determinants that are present in all strains of the virus but vary slightly from virus strain to virus strain. For example, HCV contains an antigenic determinant known as 5-1-1 (see FIG. 1). This particular antigenic determinant appears in three different forms on three different viral strains of HCV. Thus, in a preferred embodiment of the invention, all three forms of 5-1-1 appear on the multi-epitope fusion antigen used in the immunoassay of interest. Similarly, there may also be equivalent antigenic determinants from the core region of different HCV strains. Generally, equivalent antigenic determinants will have a high degree of homology with respect to the amino acid sequence, and the degree of homology when aligned is generally greater than 30%, preferably greater than 40%. A multicopy epitope of the invention can also include multiple copies that are exact copies of the same epitope.
[0079]
Representative MEFAs for use with the assays of the invention are described in International Publication No. WO 97/44469. Additional exemplary MEFAs for use herein include MEFAs called MEFA 12, MEFA 13, and MEFA 13.1. It is to be understood that these MEFAs are merely exemplary, and that other epitopes from the HCV genome may also find use with the assays of the invention and be incorporated into these or other MEFAs.
[0080]
The DNA sequence of MEFA12 and the corresponding amino acid sequence are shown in FIGS. The general structural formula of MEFA12 is shown in FIG. 6 and is as follows: hSOD-E1 (type 1) -E2 HVR consensus (type 1a) -E2 HVR consensus (types 1 and 2) -c33c short (type 1) -5-1-1 (Type 1) 5-1-1 (Type 3) -5-1-1 (Type 2)-c100 (Type 1)-NS5 (Type 1)-NS5 (Type 1)- Core (type 1 + 2) -core (type 1 + 2). This multicopy epitope comprises the following amino acid sequence numbered for HCV-1 (the amino acid sequence numbering described below is from Choo et al. (1991) Proc. Natl. Acad. Sci. USA 88: 2451). Amino acid # 1 is the first methionine encoded by the coding sequence of the core region, according to the numbering instructions provided at -2455): superoxide dismutase (SOD, which promotes recombinant expression of the protein) Amino acids 1-69; amino acids 303-320 of the polyprotein from the E1 region; amino acids 390-410 of the polyprotein representing the consensus sequence of the hypervariable region of HCV-1a E2; HCV-1 and HCV Region representing the consensus sequence of the E2 hypervariable region of -2 Amino acids 384-414 of the HCV-1 polyprotein that defines helicase; three copies of the epitope from amino acids 1689-1735 of 5-1-1 (two are HCV-1 , One from HCV-3, and one from HCV-2, these copies are equivalent antigenic determinants from three different viral strains of HCV); Amino acids 1901-1936 of the polyprotein, one HCV polypeptide C100; two exact copies of an epitope from the NS5 region of HCV-1, each of which has amino acids 2278-2313 of the HCV polyprotein; and core Two copies of three epitopes from the region (one from HCV-1 and one Is derived from HCV-2, and these copies are equivalent antigenic determinants represented by amino acids 9-53 and 64-88 of HCV-1 and amino acids 67-84 of HCV-2).
[0081]
Table 2 shows the amino acid positions of various epitopes in MEFA 12, with reference to FIGS. 7A-7F herein. The numbering in this table relates to HCV-1. Choo et al. (1991) Proc. Natl. Sci. ScL USA 88: 2451-2455. MEFAs 13 and 13.1 also share the general formula specified above for MEFA 12, with modifications as shown in Tables 3 and 4, respectively.
[0082]
[Table 2]

* The SOD protein is cleaved such that the HRP-labeled anti-SOD antibody, the detection conjugate, does not bind to MEFA. The core epitope is mutated so that the antibody against the HCV core used for detection does not bind to MEFA.
[0083]
[Table 3]

* The 5-1-1 epitope is modified by removing a potential cleavage site (CS or CA) targeted by the NS3 / 4a recombinant protein. This sequence is changed to PI instead of CS or CA. Furthermore, the SOD protein is mutated so that the detection conjugate, the HRP-labeled anti-SOD antibody, does not bind to MEFA. The core epitope is mutated such that antibodies against HCV used for detection do not bind to MEFA.
[0084]
[Table 4]

* The 5-1-1 epitope is modified by removing a potential cleavage site (CS or CA) targeted by the NS3 / 4a recombinant protein. This sequence is changed to PI instead of CS or CA. Furthermore, the SOD protein is mutated so that the detection conjugate, the HRP-labeled anti-SOD antibody, does not bind to MEFA. The core epitope is mutated so that the antibody against the HCV core used for detection does not bind to MEFA.
[0085]
In one assay format, the sample is combined with a solid support, as described further below. If the sample is infected with HCV, the core antigen and HCV antibodies to these epitopes present on the solid support will bind to the solid support components. Then, a detectably labeled anti-core antibody is added. The labeled anti-core antibody is directed against a different epitope than the anti-core antibody bound to the solid support. This anti-core antibody binds to the core antigen captured by the anti-core antibody on the solid support.
[0086]
An antigen that reacts with the captured HCV antibodies from the biological sample, the HCV antibodies of the captured samples being reactive with the NS3 / 4a epitope, is also added. This antigen is preferably an epitope from the NS3 region of the HCV polyprotein. This antigen binds to the captured HCV antibodies from the sample. Numerous antigens containing such epitopes are known, including but not limited to antigens from the c33c and c100 regions, and fusion proteins containing the NS3 epitope (eg, c25). These and other NS3 epitopes are useful in the assays of the invention and are known in the art, and are described, for example, in: Houghton et al., US Pat. No. 5,350,671; Chien et al. Proc. Natl. Acad. Sci. USA (1992) 89: 10011-10015; Chien et al. Gastroent. Hepatol. (1993) 8: S33-39; Chien et al., International Publication No. WO 93/00365; Chien, D .; Y. , International Publication No. WO 94/01778; and commonly owned U.S. patent applications Ser. Nos. 08 / 403,590 and 08 / 444,818.
[0087]
A second labeled antibody to the above antigen is added. The antibodies can be directed against any epitope contained on the antigen. For example, the antibodies can be directed against the NS3 region present in the antigen. Alternatively, if the antigen is expressed as a fusion protein, a second labeled antigen can be directed against the fusion partner. In particular, if the solid support comprises MEFA, additional antigens and antibodies can be added to the assay. These assay formats are described further below.
[0088]
A representative assay of the present invention is shown in FIG. As shown in this figure, the solid support contains two anti-core monoclonal antibodies (designated c11-3 and C11-7). These antibodies are directed against an epitope found in the N-terminal region of the core protein of amino acids 10-53 (numbered with respect to the HCV1 polyprotein sequence). This solid support also contains an epitope for NS3 / 4a. A biological sample is added to the solid support. The HCV core antigen and antibodies to the NS3 / 4a epitope, both of which are present in the sample, bind to the capture reagent on the solid support.
[0089]
Horseradish peroxidase (HRP) -labeled anti-core monoclonal antibody c11-14 (antibody to the C-terminal region of the core found at amino acids 120-130, numbered with respect to the HCV1 polyprotein sequence) is added. A fusion protein comprising a sequence from human SOD (hSOD) and an epitope from the c33c region is added, as is a second HRP-tagged antibody to the SOD portion of the fusion protein. The SOD-c33c fusion protein binds to the anti-NS3 antibody, and the anti-SOD antibody then binds to the SOD-c33c fusion protein. Detection of the label indicates the presence of an HCV infection.
[0090]
Another representative assay of the present invention is shown in FIG. The configuration of the antibody assay is an antigen-antibody-antigen sandwich capture assay using both NS3 / 4a and MEFA12. The solid support contains the two anti-core monoclonal antibodies described above, an epitope for NS3 / 4a, and a representative MEFA, MEFA12, which includes a truncated version of human SOD. As in the assays described above, a biological sample is added to the solid support. Antibodies to the HCV core antigen, as well as the NS3 / 4a and MEFA epitopes, are present in the sample, which binds to the capture reagent on the solid support. Two antigens are added, one reactive with a sample antibody that binds NS3 / 4a (as described above) and one reactive with a sample antibody that binds MEFA12. In FIG. 8, the antigen reactive with the MEFA12 / sample antibody complex is a fusion between the SOD molecule and c22ksΔ47-L44W. This c22ks antigen is derived from the core region and contains the amino acid Lys of the polyprotein. ₁₀ ~ Ser ₉₉ , As well as the normally present Arg ₄₇ And replacement of Leu at position 44 with Trp. The antigen detection conjugate is the second HRP-labeled monoclonal anti-SOD antibody described above.
[0091]
The combined antigen / antibody assay described above is particularly advantageous when both the HCV core antigen and antibodies to NS3 / 4a and / or core can be detected on the same support in the same assay. In addition, as described above, additional HCV epitopes (eg, c100, 5-1-1, SOD fusion proteins to the NS5 antigen), and proteins resulting from frameshifting in the core region of the polyprotein (eg, International Publication No. WO 99/63419) Can be used in combination cocktails that cover other nonstructural epitopes of HCV.
[0092]
For a further understanding of the invention, a more detailed discussion is provided below with respect to generating antibodies for use in the immunoassays of the invention, generating polypeptides for use in the immunoassays, and methods of performing the immunoassays. be written.
[0093]
(Generation of antibodies for use in HCV immunoassay)
As described above, the assay comprises various antibodies bound to a solid support (eg, one or more anti-core antibodies) and an antigen / antibody complex formed when HCV infection is present in the sample. Various antibodies are used to detect These antibodies may be polyclonal or monoclonal antibody preparations, monospecific antisera, human antibodies, eg, humanized antibodies, denatured antibodies, F (ab ') ₂ Antibodies such as fragments, F (ab) fragments, Fv fragments, single domain antibodies, dimeric or trimeric antibody fragment constructs, minibodies, or functional fragments thereof that bind to the antigen in question It may be a hybrid or chimeric antibody.
[0094]
Antibodies can be produced using techniques well known to those skilled in the art, as well as, for example, U.S. Patent Nos. 4,011,308; 4,722,890; 4,016,043; 3,876,504; Nos. 3,770,380; and 4,372,745. For example, polyclonal antibodies are produced by immunizing a suitable animal (eg, a mouse, rat, rabbit, sheep or goat) with an antigen of interest. To enhance immunogenicity, the antigen can be conjugated to a carrier prior to immunization. Such carriers are well-known to those skilled in the art. Immunization is generally by mixing or emulsifying the antigens in saline, preferably in an adjuvant such as Freund's complete adjuvant, and injecting the mixture or emulsion parenterally (generally subcutaneously or intramuscularly). It is done by doing. Animals are generally boosted 2-6 weeks later, preferably with one or more injections of the antigen in saline, using Freund's incomplete adjuvant. Antibodies can also be generated by in vitro immunization using methods known in the art. The polyclonal antiserum is then obtained from the immunized animal. See, for example, Houghton et al., US Pat. No. 5,350,671 for the generation of anti-HCV polyclonal antibodies.
[0095]
Monoclonal antibodies are generally prepared using the method of Kohler and Milstein (1975) Nature 256: 495-497, or a modification thereof. Typically, a mouse or rat is immunized as described above. However, instead of bleeding the animal to extract the serum, the spleen (and optionally some large lymph nodes) is removed and separated into single cells. If desired, spleen cells can be screened (after removal of non-specifically attached cells) by applying the cell suspension to a plate or well coated with the antigen. B cells (expressing a membrane-bound immunoglobulin specific for the antigen) bind to the plate and are not washed away with the rest of the suspension. The resulting B cells (or all dissociated spleen cells) are then induced to fuse with the melanoma cells to form hybridomas, and to a selective medium (eg, hypoxanthine, aminopterin, thymidine medium, "HAT"). The resulting hybridomas are plated by limiting dilution and assayed for the production of antibodies that specifically bind to the immunizing antigen (and do not bind to unrelated antigens). The selected monoclonal antibody screening hybridomas are then cultured either in vitro (eg, in tissue culture bottles or hollow fiber reactors) or in vivo (eg, as mouse ascites fluid).
[0096]
The production of various anti-HCV monoclonal antibodies is described, for example, in Houghton et al., US Pat. No. 5,350,671; Chien et al., International Publication No. WO 93/00365; co-owned patent application Ser. No. 08/403. And Kashiwakuma et al., US Pat. No. 5,871,904.
[0097]
As explained above, antibody fragments that retain the ability to recognize an antigen of interest also find use in immunoassays of interest. Numerous antibody fragments are known that contain an antigen binding site that can exhibit the immunological binding properties of an intact antibody molecule. For example, a functional antibody fragment is generated by cleaving a constant region that is not responsible for antigen binding from an antibody molecule, using, for example, pepsin, resulting in F (ab ′) 2 ₂ Generate a fragment. These fragments contain two antigen binding sites, but lack part of the constant region from each of the heavy chains. Similarly, if desired, Fab fragments (containing a single antigen-binding site) can be generated, for example, by digestion of polyclonal or monoclonal antibodies with papain. Functional fragments (containing only the variable regions of the heavy and light chains) can be produced using standard techniques, such as recombinant production of immunoglobulin molecules or preferential proteolytic cleavage. These fragments are known as Fv. See, for example, Inbar et al. (1972) Proc. Nat. Acad. Sci. USA 69: 2659-2662; Hochman et al. (1976) Biochem 15: 2706-2710; and Ehrlich et al. (1980) Biochem 19: 4091-4096.
[0098]
Single-chain Fv ("sFv" or "scFv") polypeptides are composed of V _H Coding gene and V _L Covalently linked V expressed from a gene fusion containing a coding gene _H -V _L Heterodimer. Huston et al. (1988) Proc. Nat. Acad. Sci. USA 85: 5879-5883. Chemistry that converts naturally aggregated but chemically separated light and heavy chain polypeptides from an antibody V region into sFv molecules that fold into a three-dimensional structure substantially similar to the structure of the antigen binding site A number of methods have been described for distinguishing and developing genetic structures (linkers). See, for example, U.S. Patent Nos. 5,091,513, 5,132,405, and 4,946,778. sFv molecules can be produced using methods described in the art. See, for example, Huston et al. (1988) Proc. Nat. Acad. Sci. USA 85: 5879-5883; U.S. Patent Nos. 5,091,513, 5,132,405, and 4,946,778. The design criteria involves determining an appropriate length over the distance between the C-terminus of one strand and the N-terminus of the other strand, where the linker is generally not prone to coiling Or are formed from hydrophilic low molecular weight amino acid residues that do not form a secondary structure. Such methods are known in the art. See, for example, U.S. Patent Nos. 5,091,513, 5,132,405, and 4,946,778. Suitable linkers generally include an alternative set of polypeptide chains of glycine and serine residues, and may include glutamic and lysine residues inserted to enhance solubility.
[0099]
"Mini-antibodies" or "minibodies" also find use with the present invention. Miniantibodies are sFv polypeptide chains that contain an oligomerization domain at their C-terminus, separated from the sFv by a hinge region. Pack et al. (1992) Biochem 31: 1579-1584. The oligomerization domain contains a self-associating α-helix (eg, a leucine zipper) that can be further stabilized by additional disulfide bonds. This oligomerization domain is designed to be compatible with folding in a specific direction across the membrane, and this folding is believed to facilitate in vivo folding of the polypeptide into a functional binding protein Process. Generally, miniantibodies are produced using methods well known in the art. See, e.g., Pack et al. (1992) Biochem 31: 1579-1584; Cumber et al. (1992) J Immunology 149B: 120-126.
[0100]
(Generation of antigens for use in HCV immunoassay)
As explained above, the molecules of the invention are generally produced recombinantly. Thus, polynucleotides encoding HCV antigens for use with the present invention can be made using standard techniques of molecular biology. For example, a polynucleotide sequence encoding a molecule described above can be obtained using recombinant methods (eg, by screening cDNA and genomic libraries from cells expressing the gene, or known to include the gene). By inducing the gene from a known vector). In addition, the desired gene can be isolated directly from the viral nucleic acid molecule using the techniques described in the art (eg, Houghton et al., US Pat. No. 5,350,671). The gene of interest can also be produced synthetically, rather than cloned. These molecules can be designed with the appropriate codons for a particular sequence. The complete sequence is then assembled from the overlapping oligonucleotides prepared by standard methods and assembled into the complete coding sequence. See, eg, Edge (1981) Nature 292: 756; Nambair et al. (1984) Science 223: 1299; and Jay et al. Biol. Chem. 259: 6311.
[0101]
Thus, a particular nucleotide sequence can be obtained from a vector carrying the desired sequence, or can be obtained from a variety of oligonucleotide synthesis techniques known in the art (eg, site-directed mutagenesis and polymerase chain reaction, where appropriate). PCR)) can be used to synthesize completely or partially. See, for example, Sambrook, supra. In particular, one way to obtain a nucleotide sequence encoding a desired sequence is to anneal a complementary set of overlapping synthetic oligonucleotides generated by a conventional automated polynucleotide synthesizer, and then ligate with an appropriate DNA ligase. And by amplifying this ligated nucleotide sequence with PVR. See, for example, Jayaraman et al., (1991) Proc. Natl. Acad. Sci. USA 88: 4081-4088. In addition, oligonucleotide-directed synthesis (Jones et al. (1986) Nature 54: 75-82), oligonucleotide-directed mutagenesis of existing nucleotide regions (Reichmann et al. (1988) Nature 332: 323-327, and Verhoeyen et al. (1988). ) Science 239: 1534-1536), and T ₄ Enzymatic filling-in of gapped oligonucleotides using DNA polymerase (Queen et al. (1989) Proc. Natl. Acad. Sci. USA 86: 10029-10033) is used in the present invention. Molecules with altered or increased antigen binding capacity and / or reduced immunogenicity can be provided.
[0102]
Once the coding sequence has been prepared or isolated, such a sequence can be cloned into any suitable vector or replicon. Numerous cloning vectors are known to those of skill in the art, and the selection of an appropriate cloning vector is possible. Suitable vectors include, but are not limited to, plasmids, phages, transposons, cosmids, chromosomes, or viruses that can replicate when associated with appropriate control elements.
[0103]
The coding sequence is then placed under the control of appropriate control elements, depending on the system used for expression. Thus, this coding sequence is placed under the control of a promoter, a ribosome binding site (for bacterial expression), and, if necessary, an operator, so that the DNA sequence of interest is converted into RNA by the appropriate transformant. Is transferred to The coding sequence may or may not include a signal peptide or leader sequence, which may later be removed by the host in post-translational processing. See, for example, U.S. Patent Nos. 4,431,739; 4,425,437; 4,338,397.
[0104]
In addition to control sequences, it may be desirable to add regulatory sequences that allow for the regulation of expression of the sequence in relation to the growth of the host cell. Regulatory sequences are known to those of skill in the art, and include, for example, those that cause expression of a gene that is turned on or off in response to a chemical or physical stimulus, including a regulatory compound. Other types of regulatory elements may also be present in the vector. For example, enhancer elements can be used herein to increase the level of expression of the construct. Examples include the SV40 early gene enhancer (Dijkema et al. (1985) EMBO J. 4: 761), an enhancer / promoter derived from the long terminal repeat (LTR) of Rous sarcoma virus (Gorman et al. (1982) Proc. Natl. Acad. USA 79: 6777) and elements derived from human CMV (Boshart et al. (1985) Cell 41: 521) (eg, elements contained in the CMV intron A sequence (US Pat. No. 5,688,688)). . The expression cassette further includes an origin of replication for autonomous replication in a suitable host cell, one or more selectable markers, one or more restriction sites, a potential for high copy number and a strong promoter.
[0105]
The expression vector is constructed such that the particular coding sequence is located in the vector along with the appropriate control sequences, and the position and orientation of the coding sequence relative to the control sequences is such that the coding sequence is transcribed under `` control '' of the control sequences. (Ie, RNA polymerase that binds to the control sequence DNA molecule transcribes the coding sequence). Modification of the sequence encoding the molecule of interest is desirable to achieve this purpose. For example, in some cases, it may be necessary to modify the sequence such that the sequence attaches to the control sequence in the proper orientation (ie, maintains the reading frame). Control sequences and other regulatory sequences may be ligated to the coding sequence prior to insertion into the vector. Alternatively, the coding sequence can be cloned directly into an expression vector that already contains the control sequences and the appropriate restriction sites.
[0106]
As explained above, it may be desirable to generate variants or analogs of the antigen of interest. This is especially true for NS3 / 4a. Methods for doing so are described, for example, in Dasmahapatra et al., US Patent No. 5,843,752 and Zhang et al., US Patent No. 5,990,276. The HCV protein and variants or analogs of other HCV proteins for use in the assay of interest encode the polypeptide of interest by insertion of a sequence and / or by substitution of one or more nucleotides within the sequence. Prepared by deleting a part of the sequence. Methods for altering a nucleotide sequence (eg, site-directed mutagenesis, etc.) are known in the art. See, eg, Sambrook et al., Supra; Kunkel, T .; A. (1985) Proc. Natl. Acad. Sci. USA (1985) 82: 448; Geisselsider et al. (1987) BioTechniques 5: 786; Zoller and Smith (1983) Methods Enzymol. 100: 468; Dalbie-McFarland et al. (1982) Proc. Natl. Acad. See Sci USA 79: 6409.
[0107]
The molecule can be expressed in a wide variety of systems, including insect, mammalian, bacterial, viral, and yeast systems, all of which are known in the art.
[0108]
For example, insect cell expression systems (eg, baculovirus systems) are known to those of skill in the art, and are described, for example, in Summers and Smith, Texas Agricultural Experiment Station Bulletin No. 1555 (1987). Materials and methods for baculovirus / insect cell expression systems are commercially available, especially from Invitrogen, San Diego CA ("MaxBac" kit). Similarly, bacterial and mammalian cell expression systems are well known in the art and are described, for example, in Sambrook et al., Supra. Yeast expression systems are also known in the art and are described, for example, in Yeast Genetic Engineering (Barr et al., Eds., 1989) Butterworths, London.
[0109]
Many suitable host cells for use with the above systems are also known. For example, mammalian cell lines are known in the art and include, for example, Chinese hamster ovary (CHO) cells, HeLa cells, infant hamster kidney (BHK) cells, monkey kidney cells (COS), human embryonic kidney cells, human Includes immortalized cell lines available from the American Type Culture Collection (ATCC), including but not limited to hepatocellular carcinoma cells (e.g., Hep G2), Madin-Darby bovine kidney ("MDBK") cells, and others. . Similarly, E.I. coli, Bacillus subtilis, and Streptococcus spp. Find use with the constructs of the present invention in bacterial hosts such as Particularly useful yeast host in the present invention, Saccharomyces cerevisiae, Candida albicans, Candida maltosa, Hansenula polymorpha, Kluyveromyces fragilis, Kluyveromyces lactis, Pichia guillerimondii, Pichia pastoris, include Schizosaccharomyces pombe and Yarrowia lipolytica. Insect cells for use with baculovirus expression vectors include, among others, Aedes aegypti, Autographa californica, Bombyx mori, Drosophila melanogaster, Spodoptera frugiperda and Trichopus.
[0110]
A nucleic acid molecule comprising a nucleotide sequence of interest can be stably integrated into the host cell genome or maintained on a stable episomal element in a suitable host cell using gene delivery techniques well known in the art. . See, for example, U.S. Patent No. 5,399,346.
[0111]
Depending on the expression system and host selected, the molecule is produced by growing a host cell transformed with the above expression vector under conditions in which the protein will be expressed. The expressed protein is then isolated from the host cell and purified. If the expression system secretes the protein into the growth medium, the product can be purified directly from the medium. If the product is not secreted, the product can be isolated from the cell lysate. Selection of the appropriate growth conditions and recovery methods are within the skill of the art.
[0112]
Recombinant production of various HCV antigens has been described. See, for example, Houghton et al., U.S. Patent No. 5,350,671; Chien et al. Gastroent. Hepatol. (1993) 8: S33-39; Chien et al., International Publication No. WO 93/00365; Chien, D .; Y. , International Publication No. WO 94/01778.
[0113]
(Immunodiagnostic assay)
Once generated, the anti-core antibody and NS3 / 4a antigen described above are placed on a suitable solid support for use in the present immunoassay. A solid support for the purposes of the present invention can be any material that is an insoluble matrix and can have a rigid or semi-rigid surface. Exemplary solid supports include substrates such as nitrocellulose (eg, in membrane or microtiter well form); polyvinyl chloride (eg, sheet or microtiter well); polystyrene latex (eg, beads or microtiter plates) ); Polyvinylidene fluoride; diazotized paper; nylon membrane; activated beads, magnetically responsive resin, and the like, but are not limited thereto. Specific supports include plates, pellets, disks, capillaries, hollow fibers, needles, pins, solid fibers, cellulose beads, pore-glass beads, silica gel, polystyrene beads optionally crosslinked with divinylbenzene, and grafted. Co-poly beads, polyacrylamide beads, latex beads, dimethylacrylamide beads optionally crosslinked with N-N'-bis-acryloylethylenediamine, and glass particles coated with a hydrophobic polymer.
[0114]
If desired, the molecules added to the solid support can be easily functionalized to make styrene or acrylate moieties, and therefore polystyrene, polyacrylate, or polyimide, polyacrylamide, polyethylene, polyvinyl, Of molecules to other polymers such as polydiacetylene, polyphenylene-vinylene, polypeptide, polysaccharide, polysulfone, polypyrrole, polyimidazole, polythiophene, polyester, epoxy, silica glass, silica gel, siloxane, polyphosphate, hydrogel, agarose, cellulose Enable capture.
[0115]
In one situation, the solid support is first conjugated to an HCV anti-core antibody and NS3 / 4a epitope (collectively referred to herein as a solid) under appropriate binding conditions such that the molecule is sufficiently anchored to the support. Phase components) and, optionally, one or more MEFAs. At times, anchoring to the support may be enhanced by first binding the antigen and / or antibody to a protein with better solid phase binding properties. Suitable coupling proteins include serum albumin, including bovine serum albumin (BSA), keyhole limpet hemocyanin, immunoglobulin molecules, thyroglobulin, macromolecules such as ovalbumin, and other well known to those skilled in the art. Including but not limited to proteins. Other reagents that can be used to attach the molecule to the support include polysaccharides, polylactic acid, polyglycolic acid, polymeric amino acids, amino acid copolymers, and the like. Methods for attaching such molecules to such molecules and antigens are well known to those skilled in the art. For example, Brinkley, M .; A. (1992) Bioconjugate Chem. 3: 2-13; Hashida et al. (1984) J. Am. Appl. Biochem. 6: 56-63; and Anjanyulu and Staros (1987) International J. Med. of Peptide and Protein Res. 30: 117-124.
[0116]
After reacting the solid support with the solid phase component, any non-immobilized solid phase component is removed from the support by washing, and the support binding component is then separated under appropriate binding conditions under HCV antibody and antigen ( (Referred to collectively herein as "ligand molecules"). After washing to remove any unbound ligand molecules, a second anti-core antibody directed against a different epitope than the support-bound anti-core antibody is added under appropriate binding conditions. The added anti-core antibody contains a detectable label, as described above, and acts to bind any core antigen that may be present in the sample that has reacted with the anti-core antibody bound to the support. One or more antigens that can react with antibodies present in the sample that in turn reacted with the NS3 / 4A epitope are also added. As explained above, antigens are typically derived from the NS3 region of the HCV polyprotein, and in particular, from the c33c region of HCV. For a description of the regions of the epitopes derived therefrom, see Houghton et al., US Pat. No. 5,350,671; Chien et al., Proc. Natl. Acad. See Sci (1989) 89: 10011-10015; International Publication No. WO 93/00365; and commonly owned, licensed US patent applications 08 / 403,590 and 08 / 444,818. A labeled antibody to this antigen is also added. Thus, the antibody binds to the antigen that has reacted with the anti-NS3 antibody present in the sample. To this end, the c33c epitope is conveniently provided as a fusion between c33c and human superoxide dismutase (hSOD), for example by the method described in Houghton et al., US Pat. No. 5,350,671. It can be produced recombinantly. The nucleotide and amino acid sequences of human SOD are known and are reported in Hallewell et al., US Pat. No. 5,710,033. Thus, a labeled antibody to human SOD detects the presence of a complex formed between the NS3 / 4a epitope, any antibody in the sample that reacts with this epitope, and a polypeptide that in turn binds to that antibody in the sample. Can be used to
[0117]
When the MEFA is present on a solid support, one or more additional antigens that are reactive with antibodies from a biological sample that bind to the antigen present on the MEFA can also be added to the assay. Particularly useful in this context is an antigen from the core region of HCV, and more particularly, from the c22 antigen, which comprises the 119 N-terminal core amino acid of the HCV polyprotein. One particular antigen from c22 is the amino acid Lys of the polyprotein. ₁₀ ~ Ser ₉₉ And c22ks Δ47-L44W, which contains the normally present Arg47 deletion and Leu at position 44 instead of Trp. For the c33c epitope described above, this antigen is provided as a fusion with hSOD and its labeled antibody to human SOD, and the complex formed between the antibody present in the sample and the NS3 / 4a epitope and / or MEFA (These complexes can also be used to detect the presence of HCV antigens such as c33c and c22).
[0118]
More specifically, an ELISA method can be used, wherein the wells of a microtiter plate are contacted with a solid phase component. A biological sample containing or suspected of containing the ligand molecule is then added to the coated wells. After an incubation period sufficient to allow binding of the ligand-molecule to the immobilized solid phase component, the plate can be washed to remove unbound portions and to be detectably labeled. Secondary binding molecules (labeled anti-core antibody), NS3 epitope-containing molecules, and antibodies to the NS3 epitope-containing portion are added. These molecules are reacted with any captured sample antigens and antibodies, the plates are washed, and the presence of labeled antibodies is detected using methods well known in the art.
[0119]
The assay reagents described above (including an immunoassay solid support having antibodies and antigens, and antibodies and antigens that react with the capture sample) are also provided in a kit with appropriate instructions and other necessary reagents, as described above. Immunoassays can be performed as follows. The kit may also include appropriate labels and other packaged reagents and materials (ie, wash buffers, etc.), depending on the particular immunoassay used. Standard immunoassays, such as those described above, can be performed using these kits.
[0120]
(III. Experiment)
The following is an example of a specific embodiment for implementing the present invention. This example is provided for illustrative purposes only, and is not intended to limit the scope of the invention in any way.
[0121]
Efforts have been made to ensure accuracy with respect to numbers used (eg, amounts, temperature, etc.), but some experimental errors and deviations should, of course, be allowed for.
[0122]
(Example 1)
(Combined HCV antigen / antibody immunoassay)
The HCV antigen / antibody combination immunoassay of the present invention was tested with other HCV assays to test the limits of seroconversion and to compare these limits to the limits for other commercially available assays, such as: Compared.
[0123]
(A. Materials and Methods)
(Blood sample) A commercially available panel of human blood samples was used. Such panels are available, for example, from Boston Biomedica, Inc. Bioclitical Partners, Franklin, MA (BCP); and North American Biologics, Inc., West Bridgewater, MA (BBI). , BocoRatan, FL (NABI). The days shown in Tables 5 and 6 are the days when blood was collected from the subject.
[0124]
(Monoclonal antibodies) Monoclonal antibodies c11-3, c11-7 and c11-14 were obtained from Ortho Clinical Diagnostics, Raritan, New Jersey. The c11-3 and c11-7 antibodies are directed against the N-terminal portion of the core (amino acids 10-53 by numbering for the HCV1 polypeptide). The c11-14 monoclonal antibody is directed against the C-terminal portion of the core (amino acids 120-130 by numbering for the HCV1 polypeptide). The c11-14 antibody was conjugated to horseradish peroxidase (HRP) using standard procedures.
[0125]
Monoclonal antibody 5A-3 is an anti-SOD antibody directed against amino acids 1-65 of SOD and was made using standard techniques. This antibody was conjugated to HRP as described above.
[0126]
(B. Antigen)
The c33c antigen (266 amino acids, amino acids 1192-1457 of the HCV1 polyprotein) was transformed into E. coli by the method described for the synthesis of the 5-1-1 antigen (Choo et al., Science (1989) 244: 359-362). and expressed as an internal SOD fusion polypeptide in E. coli. Recombinant antigens can be obtained from Chien et al., Proc. Natl. Acad. Sci. (1989) 89: 10011-10015. See also Houghton et al., U.S. Pat. No. 5,350,671 for procedures for producing SOD-c33c.
[0127]
The NS3 / 4a epitope used in this assay is a conformational epitope having the sequence specified in FIG.
[0128]
(C. Immunoassay format)
The Abbott PRISM assay (Abbott Laboratories, Abbott Park, IL) is a commercially available and antibody-based detection assay. The assay was performed using the manufacturer's instructions.
[0129]
The ORTHO HCV version 3.0 ELISA test system (Ortho Clinical Diagnostics, Raritan, New Jersey, referred to herein as the Ortho 3.0 assay) is an antibody-based detection assay. The assay was performed using the manufacturer's instructions.
[0130]
The Roche Amplicor assay (Roche, Pleasant, CA) is a commercially available PCR-based assay. The assay was performed using the manufacturer's instructions.
[0131]
The Gen-Probe TMA assay (San Diego, CA) is a commercially available transcription-mediated amplification assay. The assay was performed using the manufacturer's instructions.
[0132]
The Ortho Antigen Assay (Ortho Clinical Diagnostics, Raritan, New Jersey) is an antigen-based detection assay. The assay was performed using the manufacturer's instructions.
[0133]
The HCV antigen / antibody combination immunoassay was performed as follows. 4 mg / mL each of purified monoclonal antibodies C11-7 and C11-3 were combined in 1 × phosphate buffered saline (PBS), pH 7.4, and the wells were mixed. 90 ng of NS3 / 4a recombinant antigen was added to the same coating buffer. This solution was mixed for 30 minutes before coating. 200 mL of the above solution was added per well to a 96-well Costar medium binding microtiter plate (Coring, Inc.). Plates were incubated at 15-30 ° C for 16-24 hours. Plates are dH ₂ Wash twice with O, followed by 1 hour with 300 μL / well post-coating buffer (1% bovine serum albumin (BSA), 1 × PBS), and 300 μL / well of stable buffer ( The plate was washed with 1 × PBS, 1% BSA, mannitol, polyethylene glycol (PEG), gelatin) for 1 hour. The plate was aspirated and dried in a freeze dryer at 4 ° C. for 24 hours. Plates were sacheted with desiccant.
[0134]
To perform the combined antigen / antibody immunoassay, 100 μL of enhanced lysis buffer (1% N-lauryl sarcosine, 0.65 M NaCl, 50 mg / mL mouse IgG test grade (Sigma, St. Louis, MO), 1% Sulfhydryl modified BSA (Bayer), 0.1% casein) was added to the plate. Then, a 100 mL sample was added. This was incubated for 1 hour at 40 ° C. on a shaker. Plates were washed six times on an Ortho Plate Washer with 1 × PBS, 0.1% Tween-20. 200 mL of binding solution (250 ng / assay 1:75 diluted c11-14-HRP with SOD-c33c antigen and 1: 5000 diluted mouse anti-SOD-HRP (ORTHO) in HCV 3.0 sample dilution without SOD extract HCV version 3.0 ELISA test system (Ortho Clinical Diagnostics, Raritan, NJ) (all prepared 30 minutes prior to addition) The solution was incubated for 45 minutes at 40 ° C. with shaking. Washed 6 times and added 200 mL of substrate solution (1 OPD tablet / 10 mL) .OPD tablets are o-phenylenediamine dihydrochloride and hydrogen peroxide for horseradish peroxidase reaction pigmentation. Wherein, and Sigma, St.Louis, are commercially available from MO. This, for 30 minutes, and incubated in the dark at 15 to 30 ° C.. The reaction, 50 mL 4N H ₂ SO ₄ Was stopped and the plate was read at 492 nm against absorbance at 690 nm as a control.
[0135]
(D. Result)
The results of the various assays are shown in Tables 5 and 6, which show two separate experiments performed on blood samples exposed to HCV infection as indicated. The hatched area indicates detection of the virus. As shown below, the Chiron combination antigen / antibody assay detects seroconversion in all samples, while all other antibody and antigen-based assays detect seroconversion in at least one sample. That failed. In particular, none of the antibody-based assays detected seroconversion until at least day 18 (Table 5). Table 6 shows that none of the antibody-based assays detected the presence of HCV infection at day 22. In addition, the Ortho antigen-based assay failed to detect seroconversion from day 85.
[0136]
Thus, based on the above results, it is clear that the novel combination antibody / antigen assay reduces the number of false negatives obtained using other conventional antibody and antigen-based assays.
[0137]
[Table 5]

[0138]
[Table 6]

(Example 2)
(Production of NS3 / 4a conformational epitope with Thr to Pro and Ser to Ile substitutions)
The conformational epitope of NS3 / 4a was obtained as follows. This epitope has the sequence specified in FIGS. 4A-4D and differs from the native sequence at position 403 (amino acid 1428 of the HCV-1 full length sequence) and position 404 (amino acid 1429 of the HCV-1 full length sequence). In particular, the Thr that normally occurs at position 1428 of the native sequence has been mutated to Pro, and the Ser that occurs at position 1429 of the native sequence has been mutated to Ile.
[0139]
In particular, the yeast expression vector used was pBS24.1 described above. Plasmid pd. Encoding the representative NS3 / 4a epitope used in this immunoassay. hcv1a. ns3ns4aPI was made as follows. A two-step procedure was used. First, the following pieces of DNA were ligated together: (a) a 5 ′ HindIII cloning site followed by the sequence ACAAAAACAAAA, an initiator ATG, and a codon for HCV1a beginning at amino acid 1027 and continuing to the BglI site at amino acid 1046. (B) a 683 bp BglI-ClaI restriction fragment from pAcHLTns3ns4aPI (encoding amino acids 1046 to 1274); and (c) a HindIII and ClaI digested, dephosphorylated, and gel purified pSP72 vector. (Promega, Madison, WI, GenBank / EMBL accession number X65332). Plasmid pAcHLTns3ns4aPI was derived from pAcHLT, a baculovirus expression vector commercially available from BD Pharmingen (San Diego, CA). In particular, the pAcHLT EcoRI-PstI vector was prepared as well as the following fragments: 935 bp EcoRI-AlwnI corresponding to amino acids 1027-1336 of the HCV-1 genome; 247 bp AlwnI-SacII corresponding to amino acids 1364-1419 of the HCV-1 genome. A 175 bp HinfI-BglI corresponding to amino acids 1449-1509 of the HCV-1 genome; a 619 bp BglI-PstI + transcription termination codon corresponding to amino acids 1510-1711 of the HCV-1 genome. A synthetically generated 91 bp SacII-HinfI fragment corresponding to amino acids 1420 to 1448 of the HCV-1 genome and containing a PI mutation (Thr-1428 mutated to Pro, Ser-1429 mutated to Ile). The 175 bp HinfI-BglI fragment and the 619 bp BglI-PstI fragment described above were ligated and subcloned into pGEM-5Zf (+) vector digested with SacII and PstI. pGEM-5Zf (+) is commercially available from E. coli. coli vector (Promega, Madison, WI, GenBank / EMBL accession number X65308). After transformation of competent HB101 cells, mini-screening analysis of individual clones and sequence confirmation, pGEM5. The 885 bp SacII-PstI fragment from PI clone 2 was gel purified. This fragment was ligated with the EcoRI-AlwnI 935 bp fragment, AlwnI-SacII 247 bp fragment and the pAcHLT EcoRI-PstI vector described above. The resulting construct was named pAcHLTns3ns4aPI.
[0140]
The above ligation mixture was transformed into HB101 competent cells and plated on Luria agar plates containing 100 μg / ml ampicillin. Putative positives were identified by miniprep analysis of individual clones, two of which were amplified. Plasmid DNA for clones 1 and 2 of pSP72 1aHC was prepared using the Qiagen Maxiprep kit and sequenced.
[0141]
The following fragments were then ligated together: (a) a 761 bp HindIII-ClaI fragment from pSP721aHC # 1 (pSP72.1aHC was made by ligating together: pSP72 digested with HindIII and ClaI, A synthetic oligonucleotide providing a 5 'HindIII cloning site, followed by the sequence ACAAAAACAAAA, a start codon ATG and a codon for HCV1a beginning at amino acid 1027 and continuing to a BglII site at amino acid 1046, and a 683 bp BglII-ClaI from pAcHLTns3ns4aPI. A restriction fragment (encoding amino acids 1046 to 1274); (b) for the yeast hybrid promoter ADH2 / GAPDH. A 353 bp BamHI-HindIII fragment; (c) a 1320 bp ClaI-SalI fragment from pAcHLTns3ns4aPI (encoding HCV1a amino acids 1046-1711 with Thr 1428 mutated to Pro and Ser 1429 mutated to Ile); d) pBS24.1 yeast expression vector digested with BamHI and SalI, dephosphorylated and gel purified. The ligation mixture was transformed into competent HB101 and plated on Luria agar plates containing 100 μg / ml ampicillin. Miniprep analysis of individual colonies revealed the expected 3446 bp BamHI-SalI insert (ADH2 / GAPDH promoter, initiator codon ATG and HCV1a NS3 / 4a at amino acids 1027-1711 (amino acids 1-686 of FIGS. 4A-4D). (Comprising Thr 1428 (amino acid position 403 in FIGS. 4A-4D) mutated to Pro and Ser 1429 (amino acid position 404 in FIGS. 4A-4D) mutated to Ile). Was identified. This construct is called pd. HCV1a. It was named ns3ns4aPI (see FIG. 5).
[0142]
S. cerevisiae strain AD3 in pd. HCV1a. Transformation with ns3ns4aPI and one transformant was checked for expression after glucose depletion in the medium. Recombinant protein was expressed at high levels in yeast as detected by Coomassie blue staining and confirmed by immunoblot analysis using a polyclonal antibody against the helicase domain of NS3.
[0143]
(Example 3)
(Purification of NS3 / 4a conformational epitope)
The NS3 / 4a conformational epitope was purified as follows. An S. aureus derived from the above expressing the NS3 / 4a epitope. cerevisiae cells were collected as described above. The cells are suspended in lysis buffer (50 mM Tris (pH 8.0), 150 mM NaCl, 1 mM EDTA, 1 mM PMSF, 0.1 μM pepstatin, 1 μM leupeptin) and 1: 1: 1 cell: buffer: 0 Melting was performed using Dyno-Mill (Wab Willy A. Bachofon, Basel, Switzerland) or equivalent device using glass beads at a ratio of 0.5 mm glass beads. The lysate was centrifuged at 30100 × g for 30 minutes at 4 ° C., and the pellet containing the insoluble protein fraction was added to wash buffer (6 ml / g starting cell pellet weight) and rocked for 15 minutes at room temperature. The washing buffer was 50 mM NaPO ₄ (PH 8.0), 0.3 M NaCl, 5 mM β-mercaptoethanol, 10% glycerol, 0.05% octylglucoside, 1 mM EDTA, 1 mM PMSF, 0.1 μM pepstatin, 1 μM leupeptin. Cell debris was removed by centrifugation at 30100 × g for 30 minutes at 4 ° C. The supernatant was discarded and the pellet retained.
[0144]
Protein was extracted from the pellet as follows. 6 ml / g extraction buffer was added and rocked for 15 minutes at room temperature. The extraction buffer consisted of 50 mM Tris (pH 8.0), 1 M NaCl, 5 mM β-mercaptoethanol, 10% glycerol, 1 mM EDTA, 1 mM PMSF, 0.1 μM pepstatin, 1 μM leupeptin. This was centrifuged at 30100 × g for 30 minutes at 4 ° C. The supernatant was retained and ammonium sulfate was added to 17.5% using the following formula: volume of supernatant (ml) x x% ammonium sulfate / (1-x% ammonium sulfate) = to supernatant Ml of 4.1M saturated ammonium sulfate added. Ammonium sulfate was added dropwise with stirring on ice and the solution was stirred on ice for 10 minutes. The solution was centrifuged at 17700 × g for 30 minutes at 4 ° C., and the pellet was retained and stored at 2 ° C. to 8 ° C. for up to 48 hours.
[0145]
The pellet was resuspended and applied to a poly U column (Poly U Sepharose 4B, Amersham Pharmacia) at 4 ° C. as follows. The pellet was resuspended in 6 ml of poly U equilibration buffer per gram of pellet weight. The equilibration buffer consisted of 25 mM HEPES (pH 8.0), 200 mM NaCl, 5 mM DTT (freshly added), 10% glycerol, 1.2 octyl glucoside. The solution was rocked at 4 ° C. for 15 minutes and centrifuged at 31000 × g for 30 minutes at 4 ° C.
[0146]
A poly U column (1 ml resin per gram starting pellet weight) was prepared. The linear flow rate was 60 cm / hour and the packing flow rate was 133% of 60 cm / hour. The column was equilibrated with equilibration buffer and the supernatant of the resuspended ammonium sulfate pellet was loaded onto the equilibrated column. The column was washed to baseline with equilibration buffer, and the protein was eluted in one step elution in the following poly U elution buffer: 25 mM HEPES (pH 8.0), 1 M NaCl, 5 mM DTT (freshly added), 10% glycerol, 1.2 octyl glucoside. The column eluate was run on SDS-PAGE (Coomassie stained) and aliquots were frozen and stored at -80 ° C. The presence of the NS3 / 4a epitope was confirmed by Western blot using a polyclonal antibody against the NS3 protease domain and a monoclonal antibody against the 5-1-1 epitope (HCV 4a).
[0147]
In addition, protease enzyme activity was monitored during purification as follows. A sample containing the NS4A peptide (KKGSVVIVGRIVLSGKPAIIPKK) and the NS3 / 4a conformational epitope was combined with 90 μl of reaction buffer (25 mM Tris (pH 7.5), 0.15 M NaCl, 0.5 mM EDTA, 10% glycerol, 0.05 n-dodecyl). (BD-maltoside, 5 mM DTT) and mixed for 30 minutes at room temperature. 90 μl of the mixture was added to a microtiter plate (Costar, Inc., Corning, NY) and 10 μl of HCV substrate (AnaSpec, Inc., San Jose CA) was added. Plates were mixed and read on a Fluostar plate reader. The results were expressed in relative fluorescence units per minute (RFU).
[0148]
Using these methods, the product of the 1 M NaCl extract contains 3.7 RFU / min activity, the ammonium sulfate precipitate has 7.5 RFU / min activity, and the product of poly-U purification is 18. It had an activity of 5 RFU / min.
[0149]
(Example 4)
(Competitive research)
The following competition studies were performed to evaluate whether the NS3 / 4a conformational epitope detects antibodies different from other HCV antigens. Specifically, the NS3 / 4a antigen was compared to the c200 antigen as follows.
[0150]
0.5 μg and 1.0 μg of NS3 / 4a or c200 produced as described above (Hepatology (1992) 15: 19-25, ORTHO HCV Version 3.0 ELISA Test System, Ortho-Clinical Diagnostics, Radio, NJ, Radio. Was mixed with 20 μl of sample PHV914-5 (collection of initial seroconversion obtained from the blood of infected individuals) in a total volume of 220 μl (1 × PBS). This mixture was incubated for 1 hour at 37 ° C. in a microwell. The mixture was then transferred to NS3 / 4a coated plates and incubated at 37 ° C. for 1 hour. Plates were washed and assayed as follows.
[0151]
1 μg of c200 antigen was added to 10 μl of sample PHV914-5 in a total volume of about 220 μl. The mixture was incubated for 1 hour at 37 ° C in microwells, and 200 μl was transferred to NS3 / 4a coated plates (100 ng / assay) and incubated for 1 hour at 37 ° C. Plates were washed 5 times with 1 × PBS, 0.1% Tween-20. 200 μl of the binding solution (described above) was added and the plates were incubated and assayed. A control consisting of PHV914-5 and 1 × PBS (without antigen) was also treated as described above.
[0152]
Table 7 shows the results. The% inhibition result shown in the fourth column is calculated as the third column-{(second column / third column) × 100}. As can be seen, these data indicate that NS34a is neutralized by the initial seroconversion antibody and c200 is not. A strong signal was achieved when antibodies in members of the PHV914-5 c33c early seroconversion panel reacted with NS34a coated on the plate. The c200 antigen was not neutralized by these antibodies. This is shown in the upper panel of Table 7. When NS34a was mixed with the PHV914-5 sample, it was neutralized, and thus no antibody in the sample that reacted with NS34a coated on the microplate was present. These data indicate that NS34a can detect a different class of antibody than the antibody detected by c200.
[0153]
[Table 7]

(Example 5)
(Study on stability of NS3 / 4a conformational epitope)
To evaluate the role of NS3 / 4a epitope stability on the performance of the assay, the following study was performed to determine NS3 / 4a immunoreactivity over time at room temperature. Small aliquots of the stock NS3 / 4a were left at room temperature at the intervals shown in Table 8 and then frozen. All vials were coated simultaneously and tested against two initial NS3 seroconversion panels.
[0154]
As seen in Table 8, the NS3 / 4a stock is not stable and immunoreactivity decreases over time. In addition, maintaining the NS3 / 4a conformation is essential for immunoreactivity.
[0155]
Further stability studies were performed as follows. Two conformational monoclonal antibodies raised against NS3 / 4a using standard procedures were replaced with anti-HCV early seroconversion panels. Stock NS3 / 4a vials were stored at 3, 6, and 24 hour intervals at room temperature. NS3 / 4a from a frozen vial was coated at 90 ng / ml and assayed using the procedure described above. The results suggested that the two monoclonal antibodies were indeed conformational antibodies, and their reactivity was sensitive to manipulation of the stock NS3 / 4a antigen at room temperature. The reactivity of the positive control monoclonal antibody did not change.
[0156]
[Table 8]

(Example 6)
(Immunoreactivity of NS3 / 4a conformational epitope against denatured NS3 / 4a)
The immunoreactivity of the NS3 / 4a conformational epitope generated as described above was compared to NS3 / 4a denatured by adding SDS at a final concentration of 2% to the NS3 / 4a conformational epitope preparation. . Modified NS3 / 4a and conformational NS3 / 4a were coated on microtiter plates as described above. The c200 antigen (available in Hepatology (1992) 15: 19-25, ORTHO HCV Version 3.0 ELISA Test System, Ortho-Clinical Diagnostics, Raritan, New Jersey) is also a microtiter plate. The c200 antigen, which is presumed to be non-conformational due to the presence of reducing agent (DTT) and surfactant (SDS) in the formulation, was used as a comparison.
[0157]
Immunoreactivity was tested against two initial HCV seroconversion panels PHV904 and PHV914 (a human blood sample commercially available from Boston Biomedica, Inc., West Bridgewater, Mass.). Table 9 shows the results. These data suggest that denatured or linear forms of NS3 / 4a (and c200) do not detect the early seroconversion panel as early as the NS3 / 4a conformational epitope.
[0158]
[Table 9]

The immunoreactivity of the conformational epitope was also tested using a monoclonal antibody against NS3 / 4a generated using standard procedures. These monoclonal antibodies were then tested in an ELISA format against the NS3 / 4a and denatured NS3 / 4a and c200 antigens. These data indicate that the anti-NS3 / 4a monoclonal antibody reacts with NS3 / 4a and denatured NS3 / 4a in a manner similar to the seroconversion panels shown in Table 10. This result also provides further evidence that NS3 / 4a is essentially conformational if monoclonal antibodies similar in reactivity to the initial c33c seroconversion panel can be generated.
[0159]
[Table 10]

Accordingly, a novel HCV detection assay has been disclosed. From the foregoing, it is understood that while certain embodiments of the invention have been described herein for purposes of illustration, various modifications may be made without departing from the spirit and scope of the invention. You.
[Brief description of the drawings]
FIG.
FIG. 1 is a schematic representation of the HCV genome, showing various regions of the polyprotein from which the assay reagents (proteins and antibodies) of the present invention are derived.
FIG. 2
FIG. 2 is a schematic diagram of a representative antibody / antigen combination assay of the present invention.
FIG. 3
FIG. 3 shows the amino acid sequence of a representative NS3 / 4a conformational antigen for use in the assays of the invention. The bold alanine at position 182 is replaced with the native serine normally present at this position.
FIG. 4
4A-4D show the DNA and corresponding amino acid sequence of another representative NS3 / 4a conformational antigen for use in the assays of the present invention. The amino acids at positions 403 and 404 in FIGS. 4A-4D represent the substitution of Pro for Thr and the substitution of Ile for Ser in the native amino acid sequence of HCV-1.
FIG. 5
FIG. HCV1a. It is a schematic diagram of construction of ns3ns4aPI.
FIG. 6
FIG. 6 is a schematic representation of the MEFA 12.
FIG. 7
7A-7F show the DNA of MEFA 12 and the corresponding amino acid sequence.
FIG. 8
FIG. 8 is a schematic diagram of a representative immunoassay of the invention using MEFA12.

Claims (46)

An immunoassay solid support comprising at least one hepatitis C virus (HCV) anti-core antibody and at least one isolated HCV NS3 / 4a epitope bound to the support. 2. The immunoassay solid support of claim 1, comprising at least two HCV anti-core antibodies attached to the support. 2. The immunoassay solid support of claim 1, wherein the at least one anti-core antibody is directed against the N-terminal region of a HCV core antigen. 4. The immunoassay solid support of claim 3, wherein the at least one anti-core antibody is directed against amino acids 10-53 of HCV, numbered relative to the HCV1 polyprotein sequence. 2. The immunoassay solid support of claim 1, wherein said at least one anti-core antibody is a monoclonal antibody. 2. The immunoassay solid support of claim 1, wherein the NS3 / 4a epitope is a conformational epitope and comprises the amino acid sequence set forth in Figures 4A-4D. 2. The immunoassay solid support of claim 1, further comprising a multi-epitope fusion antigen bound to the support. 8. The immunoassay solid support of claim 7, wherein the multi-epitope fusion antigen comprises the amino acid sequence shown in Figures 7A-7F. An immunoassay solid support, bound to the support, two hepatitis C virus (HCV) anti-core monoclonal antibodies, and a HCV NS3 / 4a conformational epitope comprising the amino acid sequences shown in FIGS. And a support. 10. The immunoassay solid support of claim 9, wherein the two anti-core antibodies are directed against the N-terminal region of the HCV core antigen. 11. The immunoassay solid support of claim 10, wherein the two anti-core antibodies are directed against amino acids 10-53 of HCV, numbered relative to the HCV1 polyprotein sequence. An immunoassay solid support, attached to the support, two hepatitis C virus (HCV) anti-core monoclonal antibodies, a HCV @ NS3 / 4a conformational epitope comprising the amino acid sequence shown in FIGS. And a multi-epitope fusion antigen comprising the amino acid sequence shown in FIGS. 7A-7F. A method for detecting hepatitis C virus (HCV) infection in a biological sample, the method comprising:
(A) providing an immunoassay solid support according to claim 1;
(B) binding the biological sample and the solid support to the at least one anti-core antibody and the NS3 / 4a epitope, respectively, when HCV antigen and antibody are present in the biological sample; Mixing under conditions;
(C) adding the following to the solid support from step (b) under conditions that form a complex:
(I) a first detectable labeled antibody, wherein the first detectable labeled antibody is a detectable labeled HCV anti-core antibody, wherein the labeled anti-core antibody comprises: A first detectable labeled antibody directed against an HCV core epitope different from the at least one anti-core antibody bound to the solid support;
(Ii) an antigen that reacts with an HCV antibody from a biological sample that reacts with the NS3 / 4a epitope; and (iii) a second detectable labeled antibody that reacts with the antigen of (ii);
(D) detecting a complex formed between the antibody and the antigen, if any, indicating HCV infection in the biological sample;
A method comprising: The at least one anti-core antibody is directed against the N-terminal region of the HCV core antigen, and the detectable labeled HCV anti-core antibody is directed against the C-terminal region of the HCV core antigen; The method according to claim 13. The at least one anti-core antibody is directed against amino acids 10-53 of HCV, numbered against the HCV1 polyprotein sequence, and the detectable labeled HCV anti-core antibody is directed against the HCV1 polyprotein sequence. 15. The method of claim 14, wherein the method is directed against amino acids 120-130 of HCV, numbered as follows. 14. The method of claim 13, wherein the antigen that reacts with HCV antibodies from the biological sample comprises an epitope from the c33c region of the HCV polyprotein. 17. The method of claim 16, wherein said c33c epitope is fused to a human superoxide dismutase (hSOD) amino acid sequence, and wherein said second detectable labeled antibody reacts with said hSOD amino acid sequence. 14. The method of claim 13, wherein said NS3 / 4a epitope is a conformational epitope and comprises the amino acid sequence set forth in Figures 4A-4D. A method for detecting hepatitis C virus (HCV) infection in a biological sample, the method comprising:
(A) providing the immunoassay solid support of claim 2;
(B) binding a biological sample and the solid support to the at least two anti-core antibodies and the NS3 / 4a epitope, respectively, when HCV antigen and antibody are present in the biological sample; Mixing under conditions;
(C) adding the following to the solid support from step (b) under conditions that form a complex:
(I) a first detectable labeled antibody, wherein the first detectable labeled antibody is a detectable labeled HCV anti-core antibody, wherein the labeled anti-core antibody comprises: A first detectable labeled antibody directed against an HCV core epitope different from the at least two anti-core antibodies bound to the solid support;
(Ii) an epitope from the c33c region of the HCV polyprotein fused to the hSOD amino acid sequence; and (iii) a second detectable labeled antibody reactive with the hSOD amino acid sequence;
(D) detecting a complex formed between the antibody and the antigen, if any, indicating HCV infection in the biological sample;
A method comprising: 20. The method of claim 19, wherein said NS3 / 4a epitope is a conformational epitope and comprises the amino acid sequence shown in Figures 4A-4D. A method for detecting hepatitis C virus (HCV) infection in a biological sample, the method comprising:
(A) providing an immunoassay solid support according to claim 9;
(B) combining a biological sample and the solid support with the at least two anti-core antibodies and the NS3 / 4a conformational epitope, respectively, when HCV antigen and antibody are present in the biological sample. Mixing under binding conditions;
(C) adding the following to the solid support from step (b) under conditions that form a complex:
(I) a first detectable labeled antibody, wherein the first detectable labeled antibody is a detectable labeled HCV anti-core antibody, wherein the labeled anti-core antibody comprises: A first detectable labeled antibody directed against an HCV core epitope different from the at least two anti-core antibodies bound to the solid support;
(Ii) an epitope from the c33c region of the HCV polyprotein fused to the hSOD amino acid sequence; and (iii) a second detectable labeled antibody reactive with the hSOD amino acid sequence;
(D) detecting a complex formed between the antibody and the antigen, if any, indicating HCV infection in the biological sample;
A method comprising: The at least two anti-core antibodies are directed against the N-terminal region of the HCV core antigen, and the detectable labeled HCV anti-core antibody is directed against the C-terminal region of the HCV core antigen. A method according to claim 21. The at least two anti-core antibodies are directed against amino acids 10-53 of HCV, numbered relative to the HCV1 polyprotein sequence, and the detectable labeled HCV anti-core antibody is labeled with the HCV1 polyprotein sequence. 23. The method of claim 22, wherein the method is directed against amino acids 120-130 of HCV, numbered for the same. A method for detecting hepatitis C virus (HCV) infection in a biological sample, the method comprising:
(A) providing the immunoassay solid support of claim 7;
(B) combining a biological sample and the solid support with the at least one anti-core antibody, the NS3 / 4a epitope, and the multi-epitope fusion antigen when HCV antigens and antibodies are present in the biological sample. Mixing under conditions capable of binding to;
(C) adding the following to the solid support from step (b) under conditions that form a complex:
(I) a first detectable labeled antibody, wherein the first detectable labeled antibody is a detectable labeled HCV anti-core antibody, wherein the labeled anti-core antibody comprises: A first detectable labeled antibody directed against an HCV core epitope different from the at least one anti-core antibody bound to the solid support;
(Ii) a first antigen and a second antigen, wherein the antigens react with HCV antibodies from a biological sample that react with the NS3 / 4a epitope and the multi-epitope fusion antigen, respectively; And (iii) a second detectable labeled antibody that reacts with the antigen of (ii);
(D) detecting a complex formed between the antibody and the antigen, if any, indicating HCV infection in the biological sample;
A method comprising: The at least one anti-core antibody is directed against the N-terminal region of the HCV core antigen, and the first detectable labeled HCV anti-core antibody is directed against the C-terminal region of the HCV core antigen. 25. The method of claim 24, wherein the method is performed. The at least one anti-core antibody is directed against amino acids 10-53 of HCV, numbered against the HCV1 polyprotein sequence, and the detectable labeled HCV anti-core antibody is directed against the HCV1 polyprotein sequence. 26. The method of claim 25, wherein the method is directed against amino acids 120-130 of HCV, numbered as follows. 25. The method of claim 24, wherein said first antigen reactive with HCV antibodies from said biological sample comprises an epitope from the c33c region of said HCV polyprotein. 28. The method of claim 27, wherein said c33c epitope is fused to a human superoxide dismutase (hSOD) amino acid sequence, and wherein said second detectable labeled antibody reacts with said hSOD amino acid sequence. 25. The method of claim 24, wherein the second antigen that reacts with HCV antibodies from the biological sample comprises an epitope from the c22 region of the HCV polyprotein. The epitope from the c22 region includes amino acids Lys ₁₀ -Ser ₉₉ of the HCV polyprotein, numbered relative to the HCV1 polyprotein sequence, wherein the amino acids Lys ₁₀ -Ser ₉₉ are deleted at position Arg47 and at position 44. Wherein the epitope is fused to a human superoxide dismutase (hSOD) amino acid sequence, and the second detectably labeled antibody reacts with the hSOD amino acid sequence. 30. The method of claim 29. 25. The method of claim 24, wherein the multi-epitope fusion antigen comprises the amino acid sequence shown in Figures 7A-7F. A method for detecting hepatitis C virus (HCV) infection in a biological sample, the method comprising:
(A) providing an immunoassay solid support according to claim 12;
(B) combining a biological sample and the solid support with the at least two anti-core antibodies, the NS3 / 4a conformational epitope, respectively, when HCV antigens and antibodies are present in the biological sample; And mixing under conditions that allow binding to the multi-epitope fusion antigen;
(C) adding the following to the solid support from step (b) under conditions that form a complex:
(I) a first detectable labeled antibody, wherein the first detectable labeled antibody is a detectable labeled HCV anti-core antibody, wherein the labeled anti-core antibody comprises: A first detectable labeled antibody directed against an HCV core epitope different from the at least two anti-core antibodies bound to the solid support;
(Ii) an epitope from the c33c region of the HCV polyprotein fused to the hSOD amino acid sequence, and an epitope from the c22 region of the HCV polyprotein fused to the hSOD amino acid sequence; and (iii) reacting with the hSOD amino acid sequence; A second detectable labeled antibody;
(D) detecting a complex formed between the antibody and the antigen, if any, indicating HCV infection in the biological sample;
A method comprising: The at least two anti-core antibodies are directed against the N-terminal region of the HCV core antigen, and the detectable labeled HCV anti-core antibody is directed against the C-terminal region of the HCV core antigen. 33. The method according to claim 32. The at least two anti-core antibodies are directed against amino acids 10-53 of HCV, numbered against the HCV1 polyprotein sequence, and the detectable labeled HCV anti-core antibody is directed against the HCV1 polyprotein sequence. 34. The method of claim 33, directed against amino acids 120-130 of HCV, numbered as follows. The epitope from the c22 region includes amino acids Lys ₁₀ -Ser ₉₉ of the HCV polyprotein, numbered relative to the HCV1 polyprotein sequence, wherein the amino acids Lys ₁₀ -Ser ₉₉ are deleted at Arg47 and at position 44. 33. The method of claim 32, having a Trp of Leu for Leu. An immunodiagnostic test kit, comprising the immunoassay solid support according to any one of claims 1 to 12, and instructions for performing an immunodiagnostic test. A method for making an immunoassay solid support, comprising:
(A) providing a solid support; and (b) attaching at least one hepatitis C virus (HCV) anti-core antibody and at least one isolated HCV NS3 / 4a conformational epitope to the support. Binding to
A method comprising: A method for making an immunoassay solid support, comprising:
(A) providing a solid support; and (b) binding two hepatitis C virus (HCV) anti-core antibodies and an isolated HCV NS3 / 4a conformational epitope to the support. The process of
A method comprising: 39. The method of claim 37 or claim 38, further comprising binding at least one of a multi-epitope fusion antigen to the support. A multi-epitope fusion antigen, comprising the amino acid sequence shown in FIGS. 7A-7F or an amino acid sequence having at least 80% sequence identity to the amino acid sequence shown in FIGS. A multi-epitope fusion antigen that specifically reacts with anti-HCV antibodies present in a biological sample of origin. 41. The multi-epitope fusion antigen of claim 40, wherein the multi-epitope fusion antigen is at least 90% identical to the amino acid sequence shown in Figures 7A-7F or the amino acid sequence shown in Figures 7A-7F. A multi-epitope fusion antigen comprising a sexual amino acid sequence and specifically reacting with an anti-HCV antibody present in a biological sample from an individual infected with HCV. 41. The multi-epitope fusion antigen according to claim 40, wherein the multi-epitope fusion antigen has the amino acid sequence shown in Figs. 43. A polynucleotide comprising a coding sequence for a multi-epitope fusion antigen of any one of claims 40-42. A recombinant vector, comprising:
(A) the polynucleotide of claim 43;
(B) and a control element operably linked to the polynucleotide, whereby the coding sequence is capable of being transcribed and translated in a host cell.
A recombinant vector comprising: A host cell transformed with the recombinant vector according to claim 44. A method for producing a recombinant multi-epitope fusion antigen, comprising:
(A) providing a population of host cells according to claim 45; and (b) under conditions in which the multi-epitope fusion antigen encoded by the coding sequence present in the recombinant vector is expressed. Culturing a population of cells,
A method comprising:

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